key: cord-009922-t1hoox6e authors: Dearden, C. J.; Al‐Nakib, W. title: Direct detection of rhinoviruses by an enzyme‐linked immunosorbent assay date: 2005-12-07 journal: J Med Virol DOI: 10.1002/jmv.1890230211 sha: doc_id: 9922 cord_uid: t1hoox6e This paper describes the first enzyme‐linked immunosorbent assay for the detection of rhinovirus antigens in clinical specimens (nasal washings), either directly or following overnight cell culture amplification. The assay takes approximately 48 hours to perform and utilizes the same rabbit antirhinovirus hyperimmune serum as both the capture and detecting antibody. The latter has been biotin‐labelled and is detected via a streptavidin 3‐galactosidase preformed complex. This new assay has been found to be very sensitive, detecting human rhinovirus (HRV)‐EL and HRV‐2 at titres as low as 10(1.8) TCID(50) 100 μl(−1) and < 10(1) TCID(50) 100 μl(−1), respectively. Furthermore, when 57 different human rhinovirus serotypes were tested in both the HRV‐EL and HRV‐2 ELISA systems a total of 49 (86%) were found to be cross‐reactive. Of 36 clinical specimens tested by virus isolation, cell‐culture‐amplified (CCA) ELISA, and direct ELISA, 15 were positive by isolation, 11 by CCA‐ELISA, and 11 by direct ELISA. The overall correlation of the CCA and direct ELISA techniques with virus isolation was found to be 88.9% and 66.7%, respectively. The present study demonstrates that the ELISA system developed is a sensitive technique for the diagnosis of rhinovirus infections. Rhinoviruses are the major causative agent of the common cold and could be responsible for at least half of all acute respiratory infections in humans [Mogabgab, 1968; Higgins et al, 19701 . Most rhinovirus infections are trivial and self-limiting in that only a minor upper respiratory tract illness of some 2-5 days duration occurs. However, in a proportion of cases, particularly in immunocompromised individuals and children with a history of bronchitis and allergy, infection can be more serious [Craighead et al, 19691 . In addition, there is some evidence to suggest that rhinovirus infections are associated with exacerbations of bronchitis and asthma [Gregg, 19831 . Furthermore, Krilov et a1 have recently detected rhinoviruses in 9 out of 53 (17%) specimens from infants with lower respiratory tract infection [Krilov et al, 19861 . Rhinoviruses are therefore appropriate targets for treatment with antiviral chemotherapeutic agents. Rapid diagnosis of rhinovirus infection will be necessary so that antiviral treatment can be initiated. However, at present, virus isolation and identification, in an appropriate cell-culture system, may take up to 2 4 weeks to complete. We have therefore developed an enzyme-linked immunosorbent assay (ELISA) so that this time interval can be reduced to approximately 48 hours. In this test the capture and the detecting antibody are both prepared from the same rabbit hyperimmune antirhinovirus serum. The latter, however, is labelled with biotin and is then detected using a streptavidin-0-galactosidase preformed complex. This system was proven to be both sensitive and specific. In this paper we describe an assay which can be used to detect rhinoviral antigens both directly in nasal washings and following overnight cell culture amplification of the same specimen. The results obtained were compared with those yielded by conventional virus isolation procedures. The rhinovirus used to establish the ELISA procedure was the untyped human rhinovirus EL. Although a member of the rhinovirus genus according to its physical and biochemical characteristics, HRV-EL has not yet been assigned a serotype number in the collaborative scheme [Kapikian et al, 1967; Kapikian et al, 19711. HRV-EL was grown to high titre by infecting confluent monolayers of Ohio human epithelial carcinoma (HeLa) cells, in 20-oz medical flats, overlaid with 25-ml of HeLa maintenance media (BME containing 2% foetal calf serum, 0.1 3% tryptose phosphate broth, 0.088% NaH CO,, 30 mM MgCl,, and antibiotics). After the cytopathic effect had developed, the virus was released from the cells by three cycles of freezing and thawing. The tissue culture fluid containing the virus was then clarified by low-speed centrifugation (2,000 rpm for 10 minutes) and stored at -7OOC. Control antigen was prepared in the same way except that the cells were not infected. All the other rhinoviruses used in this study were passaged once from laboratory stocks. Ohio HeLa cells, grown to confluence in 25-cm2 plastic tissue culture flasks (Sterilin), were infected with virus and overlaid with 10-ml HeLa maintenance media. The tissue culture fluids were then harvested as for HRV-EL and stored at -7OOC. The titre of all the rhinovirus stocks were estimated by titration in Ohio HeLa cells. The high titre HRV-EL, prepared as an antigen, was further purified by fluorocarbon extraction. One volume of Arklone (ICI) was shaken, at room temperature, with two volumes of antigen; and the mixture was then centrifuged at 2,000 rpm for 10 minutes. The aqueous layer was harvested and centrifuged at 30,000 rpm for 2 hours. The virus pellet was then resuspended in a small volume at 0.3 M NaCl/O.1 M Tris buffer (pH 7.5) and mixed with an equal volume of Freund's incomplete adjuvant. Rabbits were immunized with the purified antigen by inoculating 0.5 ml of the emulsion intramuscularly in each hind leg followed by 1 ml intravenously 4 weeks later. The animals were bled 1 week later, and the hyperimmune serum was found to have a neutralisation titre of 1 :32,000 against 100 TCID,, of the homologous virus. A hyperimmune serum to HRV-2 was prepared following the same schedule and was found to have a neutralisation titre of 1:128 against 50 TCID,, of the homologous virus. A small aliquot of the hyperimmune serum was dialyzed overnight at 4°C against three changes of 0.1 M NaHC03 (pH 8.0) (B.D.H.). In 1 ml of dimethylsulphoxide (Sigma) 1.4 mg of biotin (N-hydroxy-succinimidobiotin) (Sigma) was dissolved. Two hundred microlitres of the biotin preparation was added to each millilitre of the hyperimmune sera, and the mixture was allowed to react for 4 hours, mixing slowly on a laboratory stirrer, at room temperature. The mixture was then dialyzed overnight at 4°C against three changes of 0.1 M phosphate-buffered saline (pH 7.2) (Difco) [Gary et al, 19831 . Eighteen volunteers inoculated intranasally with HRV-EL or saline, who took part in a separate trial at the Common Cold Unit [Zerial et al, 198 .51 provided samples for this study. Nasal washings were collected on days 2 4 after virus challenge by instilling 5 millilitres of Hanks' balanced salt solution into each nostril. The expelled fluid was mixed with an equal volume of nutrient broth and stored at -70°C. Virus in nasal washings was isolated in Ohio HeLa cells. The streptavidin wash (Amersham) was supplied as a 1 mg ml-' solution in 50 mM phosphate buffer (pH 7.4) containing 0.05% w/v sodium azide and was diluted to a working concentration (1/200) with phosphate buffered saline (ph 7.2) (Difco) for the test. The streptavidin preformed complex was supplied as streptavidin-biotinylated P-galactosidase complex (Amersham) and was diluted to a working concentration (1 /200) with phosphate buffered saline (pH 7.2) (Difco) for the test. Ortho nitrophenyl-P-D-galactosidase (ONPC) (Sigma), a water-soluble yellow product that absorbs light at 410 nm, was used as the basis of the substrate and prepared for the test as follows: 0.9 mg ml-I ONPG, 10 mM MgCl, (B.D.H.), and 0.1 M 2-mercaptoethanol (Sigma) in phosphate buffered saline (pH 7.5) (Difco) [Craven et al, 19651 . All assays were performed in rigid, nonsterile, U-bottomed, 96-well polystyrene plates (Cibco/Nunc). Wells were coated overnight at 4OC with 100 pl of rabbit hyperimmune antirhinovirus-EL serum diluted 1/4,000 with 0.05 M carbonate-bicarbonate buffer (pH 9.6) (ELISA coating buffer, Don Whitley Scientific Ltd). Plates were emptied and 100 pl of 1% v/v bovine serum albumin (BSA) (Sigma) in PBS was added for 2 hours at 37°C in order to block any nonspecific binding. The HRV-EL antigen, which had a titre of approximately lo6 TCID,, 100 pl-l, was diluted in log,, steps in PBS-Tween + 0.1% v/v BSA + 5% v/v control antigen. The control antigen (uninfected tissue culture fluid) was also diluted in the same way. Plates were washed three times with PBS-Tween (200 p1 per well) using a 12-outlet minimicrowash (Skatron) and 100 p1 of HRV-EL or control antigen was added to a set of wells and plates were then incubated overnight at 4°C. After washing as before, 100 pl of the streptavidin wash, diluted 1/200 with PBS, was added to the wells in order to block any nonspecific binding to endogenous biotin in the specimens. The plates were incubated at room temperature for 10 minutes, followed by five washes with 200 p1 per well of PBS-Tween. One hundred microlitres of biotinylated antiserum, diluted to its optimal working dilution with PBS-Tween + 0.1% v/v BSA + 5% v/v control antigen, was added and the plates were incubated at 37OC for 2 hours. After three washes as before with PBS-Tween, 100 pl of streptavidin /3-galactosidase preformed complex, diluted 1 /200 with PBS-Tween + 0.1 % v/v BSA, was added to the plates, which were again incubated at 37°C for 2 hours. Plates were then washed five times with PBS-Tween, 200 pI per well, and 100 pl of substrate (prepared approximately 20 minutes prior to use) was added to all the test wells plus a row of wells, not used in the test, as a blank. Plates were left covered at 37OC and read hourly at 410 nm using an automatic plate reader (Titertek, Multiscan, Flow Laboratories) until the colour intensity exceeded its maximum. As each dilution of HRV-EL or control antigen was added to a duplicate set of wells, the mean optical density of each set was calculated. A positive result was recorded when the mean optical density of the test wells was 1 1.5 times the mean optical density of a similar dilution of control angiten. This "cutoff" value was set after experiments were performed in which a series of negative and positive specimens at various dilutions were assayed. Mean optical densities recorded for the negative specimens never exceeded 1.5 times the mean optical density of the control antigen (undiluted), whereas the mean positive specimen optical densities were always > 1.5 times the mean control antigen optical density. A similar assay for HRV-2 was also developed using the same procedure as described for HRV-EL. The optimal dilutions of the reagents used in the ELISA systems were determined by checkerboard titrations. Experiments to determine optimal incubation times and temperatures were performed once the reagent concentrations had been standardized. Cross-reactivity experiments were performed with a total of 57 different human rhinoviruses and four other control viruses (influenza A/Eng/40/83, coronavirus 2298, echovirus 1, and coxsackie A21) plus control antigen (uninfected tissue culture fluid) as controls. The titre of all the viruses was adjusted to approximately lo4.' TCID ml-' by diluting with PBS-Tween + 0.1% v/v BSA + 5% v/v control antigen, whereas the control antigen was used undiluted in the test. The antigens were then tested in both the HRV-EL and HRV-2 ELISA systems according to the described protocol. The "cutoff" value was calculated as stated in the ELISA procedure. Therefore, a positive result was again recorded when the mean optical density (OD) of a duplicate set of sample wells was 11.5 times the mean optical density calculated for the control antigen (undiluted). Nasal washings, taken on consecutive days, from the 18 volunteers challenged with HRV-EL or saline were tested for the presence of HRV-EL antigen in the HRV-EL ELISA system. Initially a cell culture amplification procedure was employed in which 50 p1 of each nasal washing was inoculated, in duplicate, into monolayers of Ohio HeLa cells grown in microtitre plates (Nunc Microwell plate 96F with lid). After 90 minutes of adsorption at 33OC, the nasal washings were removed and 200 pl HeLa maintenance media was added to each well. The plates were incubated at 33OC overnight. After one cycle of freezing and thawing 100 pl from each test well was removed and placed in the corresponding well of a precoated ELISA plate. The plates were then incubated overnight at 4"C, and the test continued according to the ELISA protocol. Secondly, a direct ELISA system was developed in which the nasal washings or control antigen (uninfected tissue culture fluid) were added directly to each of a set of duplicate ELISA plate wells coated with either pre-or postchallenge rabbit anti HRV-EL hyperimmune serum. Plates were then incubated overnight at 4°C and the test continued as described above. Test results were calculated by subtracting the mean optical density of a specimen tested in duplicate pre-serum-coated wells from the mean optical density of the same specimen tested in duplicate post-serum-coated wells. Results for the control uninfected tissue culture fluid were also calculated in this way. A nasal washing was considered positive when the test result was 2 1.5 times that of the control uninfected tissue culture fluid. This calculation was found to be the most appropriate method of controlling the considerable variation in background optical densities obtained with different nasal wash specimens. The "cutoff" value was set after a series of positive and negative specimens had been assayed using the direct ELISA as described in the ELISA procedure. A 1% BSA block and a streptavidin wash stage were incorporated in the test in order to block unoccupied sites on the surface of the ELISA plate and endogenous biotin in clinical specimens, respectively. Experiments were performed in which each stage of the test was omitted in turn and the results compared with that obtained from a complete test. Figure 1 shows that the 1% BSA block and streptavidin wash were necessary stages in the ELISA test in order to keep background levels to a minimum. With the optimal test conditions established, experiments were performed in order to determine the limits of detection of the assay for both HRV-EL and HRV-2 antigen detection. The optical density, recorded at 410 nm, decreased with increasing dilutions of the HRV-EL and HRV-2 stocks and remained approximately the same for all dilutions of control antigen. At a titre of lo'.' TCIDso 100 pl-' and