Journal of Virological Methods, 34 (1991) 13-26 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/91/%03.50 ADONIS 0166093491004092


VIRMET 01205

Sensitive avidin-biotin amplified fluorogenic enzyme immunoassay using biotinylated monoclonal antibodies for the identification and quantitation of virus J.P. Wong, R.E. Fulton

and Y.M. Siddiqui

Biomedical Defence Section, Defence Research Establishment Suffid, Medicine Hat, Alberta, Canada (Accepted 26 March 1991)


A highly sensitive amplified fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes the high affinity interaction of the vitamin biotin for the multiple binding sites on the glycoprotein avidin, was developed for the detection and identification of a model virus, Newcastle disease virus (NDV). Monoclonal antibodies (MCA) directed against the virus were purified and labelled with biotin. Biotinylated MCA was then used with avidin-labelled enzyme and a fluorogenic substrate to detect NDV adsorbed directly on nitrocellulose membranes. Reagents were standardized and, using purified virus, the theoretical lower limit of test sensitivity of the amplified FELISA was determined to be 1 fg/ml of test sample (50 ag/well). The specificity of the amplified FELISA was evaluated by challenging the assay system with homologous and heterologous strains of NDV, and with other serologically related and unrelated viruses. The test was simple to perform and multiple samples could be conveniently assayed with results obtainable in 3-4 h. Fluorogenic Monoclonal

enzyme-linked immunosorbent assay; Avidin-biotin; Nitrocellulose; antibody; Newcastle disease virus; Detection and identification

Correspondence to: J.P. Wong, Biomedical Defense Section, Defence Research Establishment Sufield, Box 4000, Medicine Hat, Alberta, TlA 8K6, Canada.


Introduction The enzyme-linked immunosorbent assay (ELISA) is a rapid and sensitive diagnostic tool for the detection and identification of a variety of viruses (Hermann et al., 1979; Yolken and Stopa, 1979; Hildreth et al., 1982; Torrance, 1987). Though generally sensitive, conventional ELISA may not be adequately sensitive in some circumstances, for example, for the direct detection of virus antigens present in low concentration in body fluids (Chao et al., 1979; Pronovost et al., 1981; Harmon and Pawlik et al., 1982), or for environment samples where detection is typically not possible without pre-concentration of the sample (Rao et al., 1972; Hill et al., 1974). We describe an amplified fluorogenic ELISA (FELISA) with enhanced sensitivity for the detection and identification of a model virus, Newcastle disease virus (NDV). This assay utilizes biotinylated monoclonal antibody (MCA) directed against NDV to detect and identify NDV immobilized on nitrocellulose membranes. The assay takes advantage of the high affinity of biotin for the multivalent binding sites on avidin and combines the amplification effect achieved by biotin-avidin interaction with the high sensitivity of the FELISA system, described previously (Fulton et al., 1988). The amplified FELISA detected 1 fg/ml (50 ag/well) of purified virus. The test system has the added advantage that it utilizes MCA rather than polyclonal antibody (PCA) as detector antibody, thus, eliminating problems such as exhaustion of reagent supplies and batch inconsistency, often associated with the use of PCA reagents.

Materials and Methods Viruses NDV (NJ-La Sota, Bl Hitchner and NJ-Roakin), parainfluenza types 1 (Sendai), 2, 3 (HA-l) and influenza (A/PR/8/34) were purchased from American Type Culture Collection (Rockville, MD) and cultivated as described previously by Fulton et al. (1988). NDV (NJ-La Sota) was purified as previously described (Fulton et al., 1988). Labelled reagents Alkaline phosphatase-labelled affinity purified goat anti-mouse IgG was purchased from Bio-Rad Laboratories (Mississauga, Ont.). Avidin-labelled alkaline phosphatase was purchased from Sigma Chemical Co. (St. Louis, MO). MCAs specific for NDV were labelled in-house with biotin, as described below.


Monoclonal antibodies Preparation

Hybridoma clones producing MCA directed against the NJ-La Sota strain of NDV were prepared under contract by the Dept. of Immunology, University of Alberta (Edmonton, Alta.). The procedure used was that originally described by Kohler and Milstein (1975) and, briefly, was as follows. Myeloma cells (THT) (Hyclone Laboratories, Logan, UT) were fused to spleen cells derived from Balb/c mice (Charles River Ltd., St. Constant, Que.) which had previously been hyperimmunized with NDV. The fused hybrid cells were cloned and cultured in RMPI-1640 medium (Gibco/BRL, Burlington, Ont.), supplemented with 15% fetal calf serum (Gibco/BRL) and containing adenine (1.5 x 10e2 M), aminopterin (4 x lop4 M) and thymidine (3.2 x 1O-3 M) (Sigma Chemical Co.). Balb/c peritoneal cells were used as feeder cells. Culture supernatants from hybridoma clones were screened for the presence of NDV antibodies by an indirect FELISA described below. Clones which were strongly positive by FELISA were amplified by growth in RMPI-1640 medium, supplemented as described above, and then injected intraperitoneally (1 x lo6 cells/mouse) into Balb/c mice. Two weeks prior to injection, mice were primed by intraperitoneal administration of 500 ~1 of pristane (Sigma Chemical Co.) and, 24 h prior to injection, mice were irradiated at a dose of 500 rad delivered from a Gamma Cell 40 (Atomic Energy of Canada, Nepean, Ont.). Ascites fluids were harvested by paracentesis after a period of 2-4 weeks, when the peritoneal cavity of the mice had become distended. Specific antibodies to NDV in the ascites fluids were assayed by indirect FELISA. Purification MCAs were purified from mouse ascites fluids on a Bio-Gel@ high-performance hydroxylapatite (HPHT) column (Bio-Rad Laboratories) fitted to a high-performance liquid chromatography (HPLC) system (Spectral Physics, San Jose, CA). The conditions followed were those described by Juarez-Salinas et al. (1984), with the exception that total elution time was increased from 25 to 30 min and the sodium phosphate concentration in the elution buffer was increased from 300 to 350 mM. Prior to column injection, ascites fluids were clarified by centrifugation at 900 x g for 15 min followed by filtration through a 0.22 PM MillexTM-GV membrane (Millipore Corp., Mississauga, Ont.). Absorbance peaks were detected at 280 nm by a Beckman variable wavelength monitor, model 165 (Beckman Instruments Canada Inc., Toronto, Ont.) and fractions were collected manually. Fractions containing absorbance peaks were pooled and tested for antibody to NDV by indirect FELISA. Those peaks containing antibody activity were dialyzed overnight against phosphate buffered saline, pH 7.4 (PBS) and stored at -60°C until used.



gel electrophoresis

The purity of the antibody-containing peaks was documented by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was carried out using the buffer system of Laemmli (1970) on 12.5% slab gels. A constant current of 30 mA per gel was applied for approximately 4 h or until the bromophenol blue tracking dye was approximately 1 cm from the bottom of the gels. The gels were stained with Coomassie brilliant blue R-250 (Bio-Rad Laboratories). Biotinylation Four different purified MCAs were biotinylated by the procedure described by Bayer and Wilchek (1980), as follows. Dimethylformamide (Caledon Laboratories, Georgetown, Ont.) was dried over Sephadex@ G-75 beads (Pharmacia, Dorval, Que.) by adding 3.0 g of the beads to 10 ml of dimethylformamide, followed by overnight incubation at room temperature. 100 1.11of the dried dimethylformamide containing 0.5 umol of biotinyl-l\‘hydroxysuccinimide ester (Sigma Chemical Co.) was added to 10 mg of purified MCA dissolved in 1 ml of PBS. The reaction mixture was maintained at room temperature for 4 h with continuous gentle stirring and free biotin was removed by dialyzing the biotinylated product overnight at 4°C against two changes of PBS. The four biotinylated MCA were tested for immunoreactivity by the amplified FELISA, described below, and the most strongly reactive biotinylated MCA, designated as 4R2, was selected for use. Standardization

of reagents

The optimal working dilutions of biotinylated MCA to NDV and avidinlabelled alkaline phosphatase for use in the amplified FELISA were determined by checkerboard titrations in MillititerTM wells sensitized with 50 ~1 of the optimal concentration of purified virus protein (20 pg/ml; 1 pg/well). The optimal concentration of virus protein, defined as the lowest concentration required to saturate the nitrocellulose membranes, was determined experimentally by titrating with MCA directed against NDV and alkaline phosphataselabelled goat anti-mouse IgG (result not shown). Optimal dilutions of 1:300 for biotinylated MCA and 1: 10 000 for avidin-labelled alkaline phosphatase yielded the highest ratio of positive to background fluorescence and, hence, were adopted for routine use. Optimal assay conditions such as incubation time, components of washing solutions, conditions of incubation and number of washes were adopted for use from the FELISA previously described by Fulton et al. (1988).


Amplified FELISA All amplified FELISA were carried out in 96-well Millititer HA filtration plates (Millipore Corp., Mississauga, Ont.). All washing steps were as previously described (Fulton et al., 1988). Wells were first coated with test NDV by adding 50 ~1 of varying dilutions of the test sample, diluted in 0.05 M carbonate-bicarbonate buffer, pH 9.6. The plates were incubated overnight at 37°C and were then washed three times with 200 ul of PBS. Unoccupied binding sites on the membranes were blocked for 1 h at 37°C with 200 ~1 of 2% skim milk (w/v) (Alpha Milk Co., Red Deer, Alta.) in PBS containing 0.1% Tween 20 (T) (PBS-T). The plates were washed once with PBS, after which the blocking step was repeated twice. The plates were then washed three times with PBS and the optimal dilution of biotinylated MCA, 1:300, diluted in PBS-T containing 2% BSA (PBS-BSA-T), was added (50 PI/well). The plates were incubated for 1 h at 37°C and then washed five times with PBS containing 0.05% T. The wells were then incubated at 37°C for 1 h with 50 ~1 of the optimal dilution (1: 10 000) of avidin-labelled alkaline phosphatase, diluted in PBS-BSA-T. After six cycles of washing with PBS containing 0.05% T, 200 ~1 of the enzyme substrate solution, 10m4 M 4-methylumbelliferyl phosphate (4MUP) (Sigma Chemical Co.) in 10% diethanolamine buffer, pH 9.8, was added and the plates incubated for 15 min at room temperature in the dark. The fluorescent product was measured by a microFLUORTM fluorometer (Dynatech Laboratories, Alexandria, VA), as previously described (Fulton et al., 1988). The fluorometer was blanked on wells which received enzyme substrate solution only. Two sets of controls, in which PBS-BSA-T replaced virus and biotinylated MCA, respectively, were included. Test results for the amplified FELISA were considered positive if the mean of the fluorescence counts obtained was equal to or greater than two standard deviations above the mean readings in the respective control wells. Indirect FELISA Ascites fluids, hybridoma supernatants, and the fractions containing absorbance peaks from HPLC were screened for the presence of antibodies to NDV by an indirect FELISA, as follows. Wells of Millititer HA plates were coated with 50 ul of the optimal concentration of NDV, diluted in 0.05 M carbonate-bicarbonate buffer, pH 9.6. All other steps were carried out as described for the amplified FELISA, with the exception that varying dilutions of the ascites fluids, test supematants or pooled eluants from HPLC were used in place of the biotinylated MCA and alkaline phosphatase-labelled goat antimouse IgG, diluted 1:lOOOin PBS-BSA-T, was used in place of avidin-labelled alkaline phosphatase.


HA assay HA tests were performed with 0.75% rooster erythrocytes (Institute Armand Frappier, Laval, Que.) by standard techniques (Grist et al., 1974). Protein estimation Protein concentrations of immunoglobulins and of purified virus preparations were estimated by a micro-Lowry procedure (Layne, 1957). Results

Purification of MCA HPHT of MCA harvested consisted

chromatography was evaluated for its effectiveness in the purification from mouse ascites fluids. A typical chromatograph of ascites fluids, from mice injected with hybridoma clones producing MCA to NDV, of four major well-resolved absorbance peaks (Fig. 1). Assay of the 2

, 0












Fig. 1. HPHT chromatography of ascites fluids from mice injected with hybridoma clone producing MCA to NDV. Mouse ascites fluids (750 ul) were injected onto a HPHT column and the fractions were eluted by a linear gradient of 0.01 to 0.35 M sodium phosphate buffer, pH 6.8. Absorbance peaks were monitored at a wavelength of 280 mn over a total collection period of 30 min. Graphic data were obtained directly from the HPLC system output. Major peaks were numbered consecutively, 1 to 4, according to the elution time.


7lLim 1






Fig. 2. Indirect FELISA of absorbance peaks eluted from HPHT column injected with ascites fluids containing antibody to NDV. Fractions from each HPHT absorbance peak were pooled and the presence of antibodies to NDV in each peak was tested by indirect FELISA. Data points represent triplicate determinations on a single plate. Error bars are standard deviations of the mean. Negative control consisted of control ascites fluid.

pooled fractions from each of these four peaks by indirect FELISA indicated that the antibody activity to NDV was confined to peak 4 (Fig. 2). The purity of the immunoglobulin fraction in peak 4 after HPHT chromatography was confirmed by SDS-PAGE (result not shown). These results demonstrated the effectiveness of HPHT chromatography for yielding electrophoretically pure immunoglobulin fractions from crude ascites fluid. Biotinylation of MCA In order to evaluate the immunoreactivity of MCA after HPHT purification and subsequent biotinylation and to insure that biological activity had been retained after these procedures, biotinylated MCAs were titrated by amplified FELISA against a fixed (optimal) concentration of NDV immobilized on the nitrocellulose solid phase. The biotinylated MCA was highly reactive immunologically towards the immobilized NDV in the amplified FELISA and was detectable at a titer of 1:12000 (Fig. 3). Amplajkd FELISA Sensitivity The lower limit of test sensitivity of the amplified FELISA was determined by titration of varying concentrations of purified NDV (NJ-La Sota) with the optimal dilutions of biotinylated MCA and avidin-labelled alkaline phosphatase. The lowest concentration of virus protein detectable by amplified FELISA was 1 fg/ml(50 ag/well), while the range of detection was 10 pg/ml to 1 fg/ml of virus protein (Fig. 4). Specificity

The specificity of the amplified FELISA for the detection of NJ-


















Fig. 3. Immunoreactivity of biotinylated MCA by amplified FELISA. Varying dilutions (1:50 to 1:12 800) of a representative biotinylated MCA (a-0) were titrated by amplified FELISA against the optimal concentration of NDV (20 &ml) and the fluorescence counts determined. The negative control (A-A) was biotinyl-N-hydroxysuccinimide ester in place of the biotinylated MCA. Data points are the mean of triplicate determinations on a single plate. Error bars represent standard deviation of the mean. (-); Negative control + two standard deviations.

La Sota strain of NDV was investigated by challenging the assay system with homologous and several heterologous strains of NDV as well as with other serologically related and unrelated viruses. Allantoic fluids containing the equivalent of 1 and 0.1 HA units of NJ-La Sota (homologous), B 1 Hitchner and Roakin (heterologous) strains of NDV, parainfluenza types 1, 2 and 3, and influenza A/PR/8 were each assayed by amplified FELISA, and the resulting fluorescence counts compared (Fig. 5). All three strains of NDV yielded positive test results although the homologous strain (NJ-La Sota) reacted most strongly, followed in order of respective reactivity by the heterologous strains (Bl Hitchner and Roakin). Other members of the Paramyxovirus group (parainfluenza types 1, 2 and 3) were also positive but at relatively reduced fluorescence counts, compared with the homologous strain. Influenza A/PR/8, a member of the Orthomyxovirus group, did not react above the baseline control level.










Fig. 4. Sensitivity of amplified FELISA. Varying concentrations of purified NDV (10 &ml to 1 fg/ml) (lo-’ to 10-i’ g/ml) were titrated by amplified FELISA and the fluorescence counts determined. The negative control consisted of coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6) in place of purified NDV diluted in coating buffer. Data points are the mean of triplicate determinations on a single plate. Error bars represent standard deviation of the mean. (-): Negative control + two standard deviations.

El1 HA El

0 HA








0.1 HA




Fig. 5. Specificity of amplified FELISA. Infected allantoic fluids were diluted in 0.05 M carbonatebicarbonate buffer, pH 9.6, to contain 1 and 0.1 HA unit, respectively, as follows: NDV La Sota (1:32 and 1:320), NDV Bl Hitchner (1:32 and 1:320), NDV Roakin (1:512 and 1:5120), paraintluenza type 1 (1:512 and 1:5120, parainfluenza type 2 (1:4 and 1:40), paminfluenza type 3 (1:4 and 140) and influenza A (1:32 and 1:320). Each dilution was then tested by amplified FELISA. FELISA control consisted of uninfected allantoic fluid (1:4 and 1:32). FELISA titers are the mean of triplicate determinations on a single plate.


Discussion An amplified immunoassay is described which optimizes test sensitivity for NDV by combining the high protein-binding capacity of nitrocellulose as solid phase, the high sensitivity of a high-energy fluorogenic substrate, and the high affinity and multiple binding interactions of biotin and avidin. The use of nitrocellulose membrane as an adsorptive material for proteins has been well documented (Kuno and Kihara, 1967; Gilman, 1970). The capacity of nitrocellulose membrane for binding of proteins has found useful applications in Western blot techniques (Towbin et al., 1979) and in dotimmunobinding assay techniques (Hawkes et al. 1982; Dao, 1985). The major advantage in the use of nitrocellulose membrane, over other adsorptive materials, is its high binding capacity for proteins (Palfree and Elliott, 1982). This may be attributable to the large surface area available for binding, which can occur both on the membrane surface and within the membrane pore matrix. In contrast, protein binding in standard polystyrene microtiter wells can only occur on the surfaces. Nitrocellulose membrane has also been used as solid-phase support in the immunoenzymatic detection and identification of a variety of different antigens (Palfree and Elliott, 1982; Horejsi and Hilgert, 1983; Bode et al., 1984; Davis et al., 1984; Berger et al., 1985; Fulton et al., 1988). Fluorescence immunoassays have been used increasingly as an alternative to chromogenic or spectrophotometric immunoassays. This is due to the inherent greater sensitivity of fluorescence determinations compared with absorbance measurements. It is now generally accepted that a theoretical 100 to lOOO-fold increase in sensitivity can be achieved using fluorometric rather than calorimetric detection methods (Ishikawa and Kato, 1978; Shalev et al., 1980; Clark and Engvall, 1985). Fluorogenic detection methods have an additional advantage over chromogenic detection methods in that the substrate incubation time required is shorter (Shekareki et al., 1985; Yolken and Leister, 1982), thus providing for more rapid results. Fluorogenic immunoassay systems have been described for the detection and identification of a number of viruses, including rotavirus (Yolken and Stopa, 1979), herpes simplex virus (Shekareki et al., 1985), La Crosse arbovirus (Hildreth et al., 1982) and hepatitis B virus (Ishikawa and Kato, 1978; Neurath et al., 1981). Enzyme immunoassays using avidin-biotin detection systems have been described for the identification and quantitation of a number of viruses. Liu and Green (1985) used the avidin-biotin system in a two-site enzyme immunoassay for the detection of hepatitis B virus surface antigen and found a significant improvement in sensitivity over that achieved using antibody directly coupled to enzyme. Faran et al. (1986) used avidin-biotin-enzyme complex to detect Rift Valley fever virus in paraffin preparations of mosquitoes at a level not detectable by other methods. Avidin-biotin systems yield superior detectabilities and lower background levels (Liu and Green, 1985; Hahn et al., 1986) and have gained recognition as an important tool in enzyme


immunoassays, for the following reasons. Avidin has an exceptionally high affinity for biotin (Kn = lo-l4 M - ‘) (Green, 1970) biotin is easily coupled to antibodies and enzymes, often without loss of biological activity (Bayer and Wilchek, 1980), and avidin has multiple biotin-binding sites (Green et al., 1971); when biotin is covalently coupled to antibody, the biotinylated antibody can bind more than one molecule of avidin, resulting in signal amplification. MCA can be biotinylated by rendering the carboxyl group of biotin reactive towards the free amino groups of the antibody molecules. Two different esters of biotin with high reactivity towards the amino groups of proteins can be used for biotinylation: biotinyl-p-nitrophenyl (BNP) ester and biotinyl-Z6hydroxysuccinimide (B-NHS) ester. B-NHS was chosen for this study because it is more water-soluble and, therefore, easier to handle. The procedure is mild and is often used to biotinylate proteins that are biologically active (Bayer and Wilchek, 1980). The MCAs that we biotinylated using this procedure retained a high level of immunoreactivity. The methodology used in this study for the purification of MCA from mouse ascites fluids combines the unique selectivity of HPHT chromatography with the high resolving and rapid separation capabilities achieved by HPLC. Hydroxylapatite, an inorganic crystalline matrix of calcium phosphate, is capable of absorbing most proteins from solutions of low ionic strength and selectively desorbing them at higher salt concentrations. This unique characteristic of hydroxylapatite enables a single-step selective separation of MCA from the usual ascites fluid contaminants, such as albumin, transferrin and proteases. In addition, HPHT chromatography has been shown to separate MCAs that differ in light chain composition (Juarez-Salinas et al., 1984). A disadvantage in using HPHT chromatography for purification of MCA is that the immunoglobulin peak is eluted at high sodium phosphate concentration, thus extensive dialysis of the purified MCA is usually required. The theoretical lower limit of sensitivity of the amplified FELISA developed using biotin-avidin interaction for the detection of NDV was 1 fg/ml of NDV antigen (50 ag/well). This constitutes a lo-fold increase in sensitivity over the FELISA previously described by Fulton et al. (1988). In addition to its enhanced sensitivity, the biotin-avidin amplified FELISA has other advantages over the previously described FELISA. Amplified FELISA utilizes MCAs which are produced by ‘immortalized’ hybridoma cells, and hence their sources of supply are theoretically inexhaustable. On the other hand, the supply of PCA from antisera is often limited by the amount of antisera one can derive from immunized animals, depending on their size, age and immunization schedules. An additional advantage of amplified FELISA arises from the fact that MCA produced by similar clones are immunologically homologous, and will react with the antigen with constant affinity, therefore yielding more consistent results. PCA, in contrast, varies in titer, depending upon the immunization. schedule. Specificity of the biotin-avidin amplified FELISA for the NJ-La Sota strain for NDV was evaluated by challenging the assay system with several other


(heterologous) strains of NDV (Bl Hitchner and Roakin), other serotypes of the Puramyxovirus group (parainfluenza types I,2 and 3) and a member of the orthomyxovirus group (influenza A/PR/8). The observation that heterologous strains of NDV, as well as other members of the Paramyxovirus group, crossreacted in the amplified FELISA, but that influenza virus did not, is to be expected. NDV shares common structural antigens with other members of the Purumyxovirus group, but it is distinct antigenically from the orthomyxovirus group (Rhodes and Van Rooyen, 1968; Kingsbury, 1985). A highly sensitive and specific amplified FELISA, which uses nitrocellulose membranes as solid phase support, biotinylated MCA, avidin-conjugated enzyme, and a fluorogenic substrate, has been developed for the direct detection and identification of a model virus (NDV) in clinical or environmental samples. The technique detected as little as 1 fg of purified virus/ml of test sample (50 ag/well), a sensitivity which is 10 times greater than that of a previously described FELISA (Fulton et al., 1988) and lo7 times greater than that typically achieved by conventional immunoassay systems (Fulton, unpublished data). The procedures were simple to perform. Readings were obtained directly from a microprocessor-controlled microfluorometer and the procedure could easily be adapted to further automation for rapid processing of multiple samples with computer analysis of results.

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Sensitive avidin-biotin amplified fluorogenic enzyme immunoassay using biotinylated monoclonal antibodies for the identification and quantitation of virus.

A highly sensitive amplified fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes the high affinity interaction of the vitamin bioti...
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