Journal oflmmunological Methods, 25 (1979) 297--310
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© Elsevier/North-Holland Biomedical Press
A COMPARISON OF LABELLED ANTIBODY METHODS FOR THE DETECTION OF VIRUS ANTIGENS IN CELL MONOLAYERS
J.D. ORAM and A.J. CROOKS Microbiological Research Establishment, Porton Down, Salisbury, Wilts. SP4 0JG, U.K.
(Received 26 July 1978, accepted 8 August 1978)
A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-well polystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays using peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than the direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods.
INTRODUCTION L a b o r a t o r y m e t h o d s f o r t h e r a p i d diagnosis o f virus i n f e c t i o n s f r e q u e n t l y involve t h e e x a m i n a t i o n o f b i o p s y s p e c i m e n s f o r t h e p r e s e n c e o f virusspecific antigens in i n f e c t e d cells b y t h e use o f radio- or f l u o r e s c e n t - l a b e l l e d a n t i b o d i e s or, m o r e r e c e n t l y , e n z y m e - l i n k e d a n t i b o d i e s . Successful diagnosis d e p e n d s u p o n t h e p r e s e n c e o f c o m p a r a t i v e l y large a m o u n t s o f virus-specific antigens at a relatively late stage o f virus i n f e c t i o n . M u c h less a t t e n t i o n has b e e n given t o t h e d e t e c t i o n o f t h e l o w c o n c e n t r a t i o n s o f viruses w h i c h a r e p r e s e n t d u r i n g early stages o f i n f e c t i o n , in air o r w a t e r samples, or in t h e b l o o d used f o r t r a n s f u s i o n p u r p o s e s . D u e to its low antigenic mass, each virus particle b i n d s v e r y f e w a n t i b o d y m o l e c u l e s so t h a t t h e d i r e c t d e t e c t i o n o f l o w c o n c e n t r a t i o n s o f viruses is d i f f i c u l t o r i m p o s s i b l e . Using 12SI-labelled i m m u n o p u r i f i e d a n t i b o d y , S t r a n g e a n d Martin ( 1 9 7 2 ) f o u n d t h a t t h e l o w e r limit f o r t h e d i r e c t d e t e c t i o n o f c o l i p h a g e T7 was a b o u t 5 × l 0 s virions. A l m e i d a and W a t e r s o n ( 1 9 6 9 ) f o u n d , b y ' i m m u n e ' e l e c t r o n m i c r o s c o p y , a d i r e c t d e t e c t i o n l i m i t o f a b o u t 106 virions f o r s o m e a n i m a l viruses. H o w e v e r , l o w e r c o n c e n t r a t i o n s o f virus m a y b e d e t e c t e d if t h e mass o f virus antigens is a m p l i f i e d b y g r o w i n g t h e virus in a s u s c e p t i b l e h o s t cell. A n u m b e r o f labelled a n t i b o d y m e t h o d s h a v e b e e n used t o d e t e c t virus antigens in h o s t cells; t h u s F L A was used b y W h e e l o c k and T a m m ( 1 9 6 1 ) , R L A b y H a y a s h i et al. {1972), E L A b y A b e l s o n et al. {1969) and Wicker and Avra-
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meas (1969) and PAP by Dougherty et al. (1974). Although high multiplicities of infection were used in these and most subsequent studies, Carter (1969) reported the detection by the FLA method, of 100 plaque-forming units (PFU) of Semliki Forest virus (SFV) after cultivation in BHK cells for 18 h. Levitt et al. (1976) found a similar sensitivity for an indirect RLA assay used to detect Western Equine encephalomyelitis virus antigens in duck embryo cells. Despite the increasing use of host cells to produce detectable amounts of virus antigens, the quantitative aspects of the detection methods have not been fully investigated. In this investigation we have used a number of labelled antibody methods to detect virus antigens produced by SFV in primary chick embryo cells (CEC). We have attempted to define the optimal reaction conditions and have compared the sensitivities of different methods. METHODS
Virus
The A774 strain of SFV (Bradish et al., 1971) was passed once in CEC and 0.5 ml portions containing l 0 s PFU/ml were stored at --80°C; these were thawed just before use. Virus infectivity was titrated as PFU/ml in CEC suspended in agar (Bradish et al., 1971).
Preparation of antisera H y p e r i m m u n e sera against viruses were prepared in New Zealand White rabbits. For SFV, Venezuelan Equine encephalomyelitis virus (VEEV), strain TC83, and Langat virus, strain TP21, the rabbits received two intravenous injections, given 1 m o n t h apart, of 106 PFU. For influenza virus, t y p e A/WSN (HON1), several subcutaneous injections were made of about 1 mg (approximately 3 X 10 '2 virions) of purified virus emulsified in Freund's complete adjuvant. Sheep antibody to rabbit IgG fraction (SAR) was prepared b y the intramuscular injection of a sheep with 10 mg of normal rabbit IgG fraction emulsified in Freund's complete adjuvant. This was followed, at intervals of 8 weeks, by booster injections of 40 mg of IgG dissolved in saline.
Purification of antibodies The IgG fraction was prepared from normal or hyper-immune rabbit sera by the method of Sober et al. (1956). A n t i b o d y to SFV was immunopurified by the m e t h o d of Fitzgeorge and Bradish (1973). Anti-virus antibodies were titrated b y either the ring inhibition test described b y Fitzgeorge and Bradish (1973) or the haemagglutination inhibition (HI) test of Clarke and Casals (1958). SAR was purified b y adsorbing immune serum with Sepharose 4B gel coupled with a b o u t 2 mg/ml of rabbit IgG fraction. Serum (25 ml) was gently stirred with Sepharose-IgG gel (5 ml) at 37°C for 1 h and then at 4°C
299 for 16 h. The gel was washed on a Buchner funnel with phosphate-buffered saline, pH 7.4 (PBS) at 4°C until the filtrate was free of material absorbing at 280 nm and then extracted with 10 ml of 1% NaC1 + 0.1 M HC1 buffer, pH 1.2, at 0°C for 15 min. The extract was neutralized, centrifuged at 2 0 0 0 X g for 1 5 m i n to remove insoluble material and concentrated by vacuum dialysis to a b o u t 1 ml. All antibody preparations were stored, in 0.5 ml or 1 ml lots, at --20°C. Anti-virus antibody preparations were adsorbed 3 times with washed, methanol-fixed monolayers of CEC in 5 cm plastic Petri dishes. Antibody was diluted, normally 1/10, in a 2% solution of bovine gamma-globulin (BGG, fraction II, Armour Pharmaceutical Co Ltd., Eastbourne, Sussex) in PBS and 1 ml portions added to each dish which was mixed occasionally at room temperature for 2 h. Fab fragments were prepared by digestion of antibodies with papain as described by Porter (1959). The digests were dialyzed against water, with daily changes, at 4°C for 3--4 days. After centrifuging at 2000 X g for 30 min to remove the insoluble F c fragments, the Fab preparation was concentrated to about 5 mg/ml by vacuum dialysis at 0°C.
Radioactive labelling of antibody Anti-virus IgG fraction, Fab fragments and SAR were labelled with 12sI by the chloramine T method as described by Strange et al. (1971) except that each milligramme of antibody was reacted with between 2 and 4 mCi o f isotope. Preparations of 12SI-labelled antibody contained between 0.03 and 0.10 atoms of ~25I/molecule. Anti-virus IgG fraction and SAR were labelled with tritium by reaction with 3,5-3H-l-fluoro,2,4-dinitrobenzene ([3H]FDNB; 14.5 or 30 Ci/mmole) as described by Benbough and Martin (1976) except that each milligramme of antibody was reacted with from 2.5 to 5 mCi of [3H]FDNB. Preparations of [3H]anti-virus IgG fraction contained between 2.7 and 4.4 atoms of tritium/molecule and [3H]SAR contained between 10 and 35 atoms of tritium/molecule.
Conjugation of antibody with peroxidase Anti-virus IgG fraction and SAR were conjugated with horseradish peroxidase (HRP) by the m e t h o d of Nakane and Kawaoi (1974). The enzyme used was Sigma t y p e VI (Sigma London Chemical Co. Ltd., Poole, Dorset) with Rz values between 2.8 and 3.2. Conjugates were purified b y filtration through a column (96 mm X 1.6 cm) of Ultragel AcA-44 (LKB Instruments Ltd., South Croydon, Surrey), equilibrated with PBS at 4°C, with a flow rate o f 20 ml/h. The column was calibrated with proteins of known mol. wt. and fractions corresponding to a 1 + 1 conjugate of HRP and IgG (mol. wt. about 190,000) were pooled and stored, at a concentration of about 100 ~g/ ml, in 1 ml lots at --20°C.
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Peroxidase-antiperoxidase complexes (PAP) A preparation was kindly provided by Dr. R.E. Strange of this establishment. It was prepared as described by Sternberger (1974) and contained 3 mg/ml of protein and had a peroxidase : antiperoxidase mole ratio of 1.42 : 1.
Fluorescent labelling of antibody SAI~ was reacted with powdered fluorescein iso-thiocyanate (FITC, isomer 1, B.D.H., Poole, Dorset) as described by Goldman (1968) and untreated FITC removed by filtering the reaction mixture through a column (20 cm × 0.9 cm) of Sephadex G-50 equilibrated with PBS. Fluorescein-SAR (F-SAR) conjugates contained about 3 mg/ml of protein and had fluorescein : protein mole ratios of between 2.1 : 1 and 3.5 : 1. For use it was diluted 1/100 in a 0.25% solution of Evans blue stain in a mixture of foetal calf serum plus PBS (1 + 1).
Detection of virus antigens in cell monolayers Monolayers of CEC were prepared in 96-well polystyrene tissue culture plates (Microtest II, Cat. No. 3040, from Falcon, Oxnard, CA, U.S.A.). Suspensions of CEC in medium (medium 199 plus 10% foetal calf serum and 0.088% NaHCO3) were adjusted to about 106 cells/ml and 0.1 ml added to each well, the plates covered with Microtest film (Falcon) and incubated overnight at 37°C in an atmosphere of 5% CO2 plus 95% air. The confluent monolayers were infected with 0.1 ml of dilutions of virus or mock-infected with medium and incubated for various times at 37°C. The cultures were washed with a 1% solution in PBS of bovine plasma albumin (fraction V, Armour Pharmaceutical Co. Ltd., Eastbourne, Sussex), followed by PBS and then fixed with methanol as described by Robb (1973), except that the methanol was precooled to --20°C. Finally, the fixed cultures were dried in a stream of air. Before reaction with antibody, the fixed cultures were rinsed with a 2% solution of BGG in PBS. Antibodies were diluted in 2% BGG solution and filtered, just before use, through a Millipore filter (0.45 t~m) and 25 t~l portions added from a Pasteur pipette to each culture. After incubation at room temperature, usually for 2 h, excess antibody was shaken from the plate which was then washed 4 or 5 times with a 1% solution in PBS of Brij 35 (polyoxyethylene glycol, B.D.H., Poole, Dorset). Cultures reacted with RLA were extracted with 0.1 ml of 0.1% Brij 35 plus 0.2 M acetic acid for 30 min at room temperature (K.L. Martin, personal communication). The radioactivity of samples containing 125I-labelled antibodies was measured in a well-type sodium iodide crystal scintillation counter (automatic spectrometer, NE8311, Nuclear Enterprises, Edinburgh) with a counting efficiency of about 40%. Samples containing tritiated antibody were mixed with 20 vol of Bray's scintillation fluid and the radioactivity measured in a Philips liquid scintillation counter (model PW 4510; N.W.
301
Philips Gloeilampenfabrieken, Eindhoven, The Netherlands), with a counting efficiency for tritium of about 40%. Cultures reacted with ELA or PAP were incubated with 200 ~l of peroxidase reagent and examined after 1 h at room temperature. The reagent was a solution of 80 mg of 5-aminosalicyclic acid in 100 ml of 0.05 M sodium acetate buffer, p H 6 . 0 ; after decolorizing with active charcoal, 0.1 vol of 30% (w/v) H202 were added just before use. Normally, the plates were examined by eye although in a few experiments the contents of the wells were transferred to a spectrophotometer and measured at 450 nm. Both methods gave very similar results. Cultures stained with FLA were wetted with 1 drop of a mixture of glycerol plus PBS (1 + 1) and the inverted plates examined with a Vickers M41 Photoplan fluorescence microscope (Vickers Ltd., York). Incident lighting was used from an Osram HBO 200 mercury vapour lamp with a BG 12/2 mm exciter filter and a combination of filters (OG 1/1.5 mm and GG 9/1.5 mm) as a barrier filter. The cultures were scanned, using 10 X oculars and a 10 X objective, and those showing specifically fluorescing multicellular loci scored as virus-positive. RESULTS
Direct assays using RIA The o p t i m u m reaction conditions for the detection of SFV in monolayer cultures of CEC were investigated using 12SI-labelled preparations of immunopurified antibody, the IgG fraction from antisera and Fab fragments derived from the IgG fraction. Since the different antibody preparations gave very similar results, only those obtained with the IgG fraction are presented in detail. For convenience, some results are expressed as virus/control signal ratios, defined as: mean counts/min from infected cultures/mean counts/min from mock-infected cultures. The o p t i m u m time for the reaction of infected monolayers with labelled antibody was about 2 h (Fig. 1), longer reaction times resulted in higher backgrounds and lower virus/control signal ratios. A reaction time of 2 h was used in all subsequent work with both direct and indirect antibody methods. The a m o u n t of radioactivity bound by infected cultures were directly proportional to the concentration of labelled antibody (Fig. 2). Labelled antib o d y was normally used at a concentration of about 10 ~g/ml (HI titre of about 2). This gave relatively high signals and a reasonable e c o n o m y in labelled antibody. The counts from mock-infected cultures were only 2--3 times higher than the instrument background; this had the important practical advantage that signals from virus antigens could be recognised with some confidence. A threshold signal was established, for each Microtest plate, above which individual cultures were regarded as virus-positive but which excluded virtually all the mock-infected cultures. Thresholds based on the mean plus either 2 or 3 standard deviations of mock-infected cultures
302
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Fig. 1. Rate of uptake of 12SI-labelled anti-SFV IgG fraction by monolayers of CEC. Washed and fixed cultures (n = 8) were reacted with 12SI-labelled anti-SFV IgG (128 pg/ ml, 3.9 × 107 counts/min/ml) for the times shown. ©, mean counts/min from cultures infected with 100 PFU of SFV and incubated for 18 h; e, mean counts/min from mockinfected cultures; bars indicate standard deviations.
excluded about 95% or 100%, respectively, of the mock-infected cultures. Our results are based on a threshold of the mean plus 3 standard deviations. The sensitivity of the m e t h o d was determined after various times of virus replication. Results are expressed as detection limits, defined as the infectivity of the input virus (PFU/culture) required to produce virus-positive signals in 50% of the cultures. Detection limits were obtained by plotting the percentage of virus-positive cultures versus the infectivity of the inoculum for each incubation time. This gave approximately parallel plots (Fig. 3), with good agreement between replicates on different days. A plot of the derived detection limits versus time of incubation (Fig. 4) shows that the m e t h o d was relatively insensitive after short incubation periods, but was very sensitive after overnight replication of virus. Thus, after 8 h incubation, the detection limit was about 700 PFU/culture, or 7 × 103 PFU/ml in the inoculum, but increasing the incubation time of the cultures to 16 h resulted in a 100fold increase in sensitivity, with a detection limit of about 8 PFU/culture. Results obtained using all-labelled anti-SFV IgG fraction were very similar to those given by 12SI-labelled antibody except that lower radioactive signals were obtained, even though the radioactivities (counts/min/pg of protein) of the two preparations were similar. Since the signals from mock-infected cultures were n o t significantly higher than the instrument background, nonspecific adsorption of antibody was negligible. The sensitivity of the m e t h o d
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(Fig. 4) was equal to that obtained using 12SI-labelled antibody except that no virus was detected at 8 h (highest inoculum: 2 0 0 0 PFU/culture). Hence, for all but the shortest periods o f virus replication, 3H-labelled antibody, which stored well at --20°C, was as effective as the 12SI-labelled preparations.
Direct assays using ELA The o p t i m u m conditions for the use o f anti-virus IgG-HRP conjugate were similar to those determined for direct RLA, except that high concentrations o f conjugate (above 50 gg/ml) gave false-positive reactions, presumably due to non-specific adsorption o f antibody. D e t e c t i o n limits obtained using a conjugate concentration o f 30 ~g/ml are given in Fig. 4. Similar values were obtained, using several batches o f conjugate. The m e t h o d was about half as sensitive as the direct RLA m e t h o d for the detection o f virus antigens in CEC.
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Fig. 3. Detection of SFV antigens in CEC by direct assays using RLA. CEC monolayers (n = 20) were infected with SFV and incubated for the following times; o, 8 h; ~, 12 h; D, 16 h; v, 18 h; e, 24 h; A, 40 h. Washed and fixed cultures were reacted with 12sIlabelled anti-SFV IgG, 2.5 pg/ml, 1.9 X 106 counts/min/ml. Cultures giving radioactive signals higher than the mean plus 3 standard deviations of the counts from mock-infected cultures (n = 16) were scored as virus-positive.
Detection o f virus antigens by indirect assays T h e m a i n a d v a n t a g e o f i n d i r e c t a n t i b o d y m e t h o d s is t h a t a single t y p e o f l a b e l l e d a n t i b o d y ( a n t i - I g G ) c a n be u s e d t o d e t e c t a v a r i e t y o f a n t i g e n s . S i n c e it is f r e q u e n t l y a s s e r t e d t h a t i n d i r e c t a n t i b o d y m e t h o d s are m o r e s e n s i t i v e t h a n d i r e c t p r o c e d u r e s , we h a v e c o m p a r e d t h e s e n s i t i v i t i e s o f a n u m b e r o f i n d i r e c t assays w i t h t h o s e o f t h e d i r e c t m e t h o d s .
Indirect assays using 3H-labelled S A R S i n c e p r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t t h e b a c k g r o u n d signals w e r e m u c h h i g h e r t h a n t h o s e o b t a i n e d b y d i r e c t R L A m e t h o d s , we i n v e s t i g a t e d several
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Fig. 4. Detection limits for SFV by labelled antibody methods. Each point, derived from curves of the type shown in Fig. 3, represents the infectivity of the input virus required to give virus-positive signals in 50% of the cultures, o, 12SI-labelled anti-SFV IgG (n = 20); e, 3H-labelled anti-SFV IgG (n --- 20); ~, HRP-labelled anti-SFV IgG (n = 24); A, anti-SFV IgG followed by HRP-labelled SAR (n = 24); El, anti-SFV IgG followed by 3H-labelled SAR (n = 60); I , PAP (n = 12); ~, anti-SFV IgG followed by fluorescein-labelled SAR (n = 60).
reaction parameters in an attempt to minimise non-specific adsorption of antibody. Immunopurified anti-virus antibody, the IgG fraction from anti-virus antiserum and Fab fragments derived from the IgG fraction were each adsorbed 3 times with CEC and tested, at approximately equal concentrations, with cultures incubated with or without 100 PFU of SFV for 18 h. As expected, immunopurified antibody gave the best results with virus/control signal ratios, in replicate experiments, of 12.2 and 15.4. The IgG fraction gave virus/control signal ratios of 7.8 and 10.1 and unfractionated antiserum gave ratios of 3.9 and 4.1. Although Fab fragments gave low background counts, the signals from infected cultures were much lower than those given b y undigested antibody molecules (virus/control signal ratios 1.7 and 2.3), indicating that the F c portion of the anti-virus antibody molecule was important for the binding of [3H] SAR. Whereas in the direct RLA m e t h o d the virus/control signal ratios were directly related to antibody concentration, the use of high concentrations of
306
anti-virus antibody in the indirect method resulted in very high non-specific adsorption of antibody and low virus/control signal ratios (Fig. 5). In some experiments virus/control signal ratios of about 1 were obtained at high concentrations of antibody (1--2 mg/ml). The highest ratios were obtained using anti-SFV IgG concentrations of between 10 and 20 ~g/ml (HI titres of about 2--4). In most subsequent experiments anti-virus antibody preparations were used at an HI titre of about 2. High concentrations of immunopurified antivirus antibody also gave reduced virus/control signal ratios, although the effect was less marked than with IgG preparations. The effect of [3H] SAR concentration was determined with cultures which had been reacted with anti-virus IgG fraction at a concentration of 20 ~g/ml (HI titre -- 4). The highest virus/control signal ratio was obtained with a [3H]SAR concentration of about 10 ~g/ml; concentrations of [3H]SAR below about 5 gg/ml resulted in lower sensitivities. The sensitivity of the indirect RLA method using [3H]SAR was 2--5-fold
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Fig. 5. T i t r a t i o n o f a n t i - S F V IgG fraction in indirect assays using R L A . Washed and f i x e d cultures were reacted w i t h a n t i - S F V IgG at the d i l u t i o n s s h o w n ( u n d i l u t e d c o n c e n t r a t i o n , 1 5 . 2 m g / m l , HI titre, 8 0 0 0 ) , f o l l o w e d b y [ 3 H ] S A R at 1 3 . 5 pg/ml, 2.3 × 10 ? c o u n t s / r a i n / ml. o, m e a n o f c o u n t s / m i n f r o m cultures (n = 8) i n f e c t e d w i t h 1 0 0 P F U and i n c u b a t e d 18 h; bars indicate standard deviations; o, m e a n o f c o u n t s / m i n from m o c k - i n f e c t e d cultures (n = 8); A, virus/control signal ratios.
307 lower than that obtained using the direct R L A methods (Fig. 4). For example, the detection limits after cultivation of SFV for 16 h were a b o u t 8 and 40 PFU/culture for the direct and indirect R L A methods, respectively. The lower sensitivity of the indirect method appeared to be due partly to higher non-specific adsorption of antibody. At relatively short incubation times (8--12 h) there was a good correlation between the infectivity of the inoculum and the radioactive signal. However, no correlation was found after longer incubation periods, presumably since these allowed sufficient time for the synthesis of approximately equal amounts of virus antigen in all infected cultures. The specificity of the m e t h o d was examined by reacting SFV-infected cultures with normal rabbit serum, antibody to SFV or hyperimmune sera to the serologically unrelated VEEV, Langat virus or influenza virus. Only the anti-SFV antibody produced virus-positive signals. Cultures infected with VEEV, Langat virus or influenza virus and reacted with anti-SFV antibody followed by [ 3H] SAR gave virus-negative signals. The validity of results obtained b y the indirect assay was tested b y relating the radioactive signals obtained from individual cultures to the infectivities of the corresponding culture supernatant fluids. Cultures were infected with between 1.7 and 150 P F U and after incubation for 40 h the supernatant fluids were removed for infectivity titrations and the monolayers reacted with anti-virus antibody followed by [3H]SAR. Virus infectivity was detected in 39 of the 60 cultures tested, with virus yields between 3 × 104 and 1.4 X l 0 s PFU/culture (assay limit, 10 PFU/culture). With the exception of the two lowest-yielding cultures (3.4 X 104 PFU and 5 X 104 PFU) all the virus-producing cultures gave radioactive signals (range, 6529-17,179 counts/min) well above those (mean = 3200 counts/min; S.D. = 592) obtained from the 32 uninfected cultures. None of the virus-negative cultures gave a signal higher than the control values. Indirect assay using 1~SI-labelled S A R The o p t i m u m concentrations of anti-SFC antibody and ~25I-labelled SAR were very similar to those determined using [3H]SAR. The method was a b o u t as sensitive as the direct R L A method and rather more sensitive than the indirect R L A m e t h o d using [3H]SAR, possibly because the 12SI-label had a higher specific activity. Indirect assay using E L A The best discrimination between virus-infected and mock-infected cultures, and hence the highest sensitivity, was obtained using a b o u t 5 pg/ml of anti-virus IgG fraction followed by 5--10 pg of SAR-HRP conjugate, each for 2 h at room temperature. In a limited study, similar results were obtained using twice the concentrations of antibody and conjugate for a 1 h reaction time. The sensitivity of the m e t h o d was a b o u t the same as the direct ELA
308 m e t h o d and slightly higher than the indirect RLA m e t h o d using [3H]SAR (Fig. 4). Indirect assay using PAP As with the ELA m e t h o d , close attention to reaction conditions was essential, otherwise the mock-infected cultures gave non-specific colour reactions. Best results were obtained using anti-virus IgG fraction at a concentration of about 5 pg/ml followed by SAR at about 10 ~g/ml and then PAP at about 3 pg/ml, each for 2 h at room temperature. The m e t h o d was approximately twice as sensitive as the ELA m e t h o d and about as sensitive as the direct RLA m e t h o d (Fig. 4). Indirect assays using F L A The o p t i m u m reaction conditions were similar to those determined for other indirect methods. Immunopurified anti-virus antibody or anti-virus IgG fraction were used at concentrations of 5--10 gg/ml followed by F-SAR at about 30 t~g/ml. The detection limits (Fig. 4) were very similar to those obtained using [3H]SAR. Although Carter (1969) observed non-specific fluorescence in CEC, this was not a problem in this investigation in which we adsorbed the antibody with CEC and used Evans blue as a counterstain. DISCUSSION The sensitivity of a m e t h o d for the detection of virus antigens in monolayers of host cells depends on a number of factors including the efficiency of uptake of virus, the rate of virus replication and the efficiency of the detection system. Clearly, the efficiency of virus uptake and rate of virus replication is influenced by the choice of host cell and the cultural conditions. The efficiency of the detection system depends on the purity and avidity of antibody preparations, the extent of labelling of the antibody and the sensitivity of the m e t h o d used to detect the label. The effect of antibody purity was examined by the indirect RLA method. The virus/control signal ratios obtained using IgG preparations were about twice those given by unfractionated antiserum and there appeared to be little further advantage in using immunopurified antibody, the preparation of which requires relatively large amounts of antigen for use as an immunoadsorbent. We found that the sensitivities of some methods, especially indirect assays with RLA and ELA, were to some extent limited by the level of the background signals from uninfected cultures. Non-specific binding of anti-virus antibody appeared to be the most important factor and was considerably reduced by multiple adsorptions of antibody with host cells. It was further reduced by careful selection of the concentration of anti-virus antibody, previously found by Schieble and Cottam (1977) to be important in the detection of influenza virus antigens by the indirect RLA method. The back-
309 ground signals were also found to depend on the concentration of the second antibody (3H- or 125I-labelled SAR), with an optimum concentration (40 pg/ ml; 250 ng/well) very similar to that reported by Hutchinson and Ziegler (1974). However, using optimum reaction conditions for the indirect R L A method, the background signals were still 2--3-fold higher than those obtained in the direct method, which was more rapid to perform. In the direct R L A method, the highest virus/control signal ratios were obtained at relatively high concentrations of anti-virus antibody. These results appear to contradict the predictions of Kalmakoff et al. (1977) that the highest sensitivity is obtained using minimal amounts of labelled antibody. We confirm that the percentage of bound antibody falls with increase in labelled antibody concentration, although in our experiments the fall was small, i.e. from 0.25% at 5.9 ng/well to 0.14% at 740 ng/well. However, the most important factor, given low background signals, would appear to be the total amount, rather than percentage, of b o u n d antibody and we found that this was 40-fold higher, with only a 5-fold increase in the background signal, at the higher concentration of labelled antibody. We have used the m e t h o d of Benbough and Martin (1976) to tritiate antibodies and confirm that [3H]FDNB is a very effective labelling reagent and that tritiated antibodies can be stored for months at --20°C, without loss of serological reactivity. Although [3H]FDNB is a relatively expensive reagent, its use eliminates the need to radio-iodinate antibody at frequent intervals, with the attendant radiochemical hazard to health. Although the ELA m e t h o d has been used to detect or locate a variety of viruses in infected cells, most investigators have used high multiplicities of virus. We have found that low concentrations of virus can be detected by this method, provided that the reaction conditions were carefully controlled. The main disadvantage of both ELA and PAP methods was that antibodies and conjugates appeared to bind, non-specifically, to host cells so that background readings were much higher than in sandwich-antibody assays. Although cultures producing high yields of virus gave unambiguous results, it was more difficult to discriminate between low-yielding and uninfected cultures. Nevertheless, direct and indirect assays using ELA proved to be reliable and sensitive. The direct assay using ELA was especially easy to perform and was the most rapid of the methods examined. Although the PAP method was very sensitive, the assays were relatively slow and tedious to perform. We found it difficult to complete the PAP assays within a normal working day, although the reaction times could probably be shortened with little loss in sensitivity. Microplate assays with ELA are well suited to automation (Ruitenberg et al., 1976) and to full containment for use with dangerous pathogens. Results obtained b y each method were reproducible and consistent between batches of virus or labelled antibody. We conclude that the best method, of those which we have examined, for the detection of a single or limited number of types of virus would be direct assays using ELA or
310 R L A , using tritiated a n t i b o d y . Where an indirect m e t h o d is preferred, indirect assays using E L A a p p e a r e d t o be slightly m o r e sensitive and easier to p e r f o r m t h a n t h e o t h e r indirect m e t h o d s . ACKNOWLEDGEMENTS We wish to t h a n k C.J. Bradish and R.B. F i t z g e o r g e f o r supplies o f virus and antisera, R.E. Strange for p r o v i d i n g a sample o f PAP and these and o t h e r colleagues f o r m u c h advice and c o m m e n t . REFERENCES Abelson, H.T., G.H. Smith, H.A. Hoffmann and W.P. Rowe, 1969, J. Natl. Cancer Inst. 42,497. Almeida, J.D. and A.P. Waterson, 1969, Adv. Virus Res. 15,307. Benbough, J.E. and K.L. Martin, 1976, J. Appl. Bacteriol. 41, 47. Bradish, C.J., K. Allner and H.B. Maber, 1971, J. Gen. Virol. 12,141. Carter, G.B., 1969, J. Gen. Virol. 4,139. Clarke, D.H. and J. Casals, 1958, Am. J. Trop. Med. Hyg. 7,561. Dougherty, R.M., H.S. DiStefano and A.A. Marucci, 1974, in: Viral Immunodiagnosis, eds. E. Kurstak and R. Morisett (Academic Press, New York) p. 89. Fitzgeorge, R.B. and C.J. Bradish, 1973, Immunochemistry 10, 21. Goldman, M., 1968, Fluorescent Antibody Methods (Academic Press, New York) p. 101. Hayashi, K.J., J. Rosenthal and H.L. Notkins, 1972, Science 176,516. Hutchinson, H.D. and D.W. Ziegler, 1974, Appl. Microbiol. 28,935. Kalmakoff, J., A.J. Parkinson, A.M. Crawford and B.R.G. Williams, 1977, J. Immunol. Methods 14, 73. Levitt, N.H., H.V. Miller and G.A. Eddy, 1976, J. Clin. Microbiol. 4, 382. Nakane, P.K. and A. Kawaoi, 1974, J. Histochem. Cytochem. 22, 1084. Porter, R.R., 1959, Biochem. J. 73,119. Robb, J.A., 1973, in: Tissue Culture Methods and Application, eds. P.F. Kruse and M.K. Patterson (Academic Press, New York) p. 517. Ruitenberg, E.J., B.M.J. Brosi and P.A. Steerenberg, 1976, J. Clin. Microbiol. 3,541. Schieble, J.H. and D. Cottam, 1977, Infect. Immun. 15, 66. Sober, H.A., F.J. Gutter, M.M. Wyckoff and E.A. Peterson, 1956, J. Am. Chem. Soc. 78,756. Sternberger, L.A., 1974, Immunocytochemistry (Prentice-Hall, Englewood Cliffs, NJ) p. 219. Strange, R.E. and K.L. Martin, 1972, J. Gen. Microbiol. 72,127. Strange, R.E., E.O. Powell and T.W. Pearce, 1971, J. Gen. Microbiol. 67,349. Wheelock, E.F. and I. Tamm, 1961, J. Exp. Med. 113,301. Wicker, R. and S. Avrameas, 1969, J. Gen. Virol. 4,465.
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© Elsevier/North-Holland Biomedical Press
A COMPARISON OF LABELLED ANTIBODY METHODS FOR THE DETECTION OF VIRUS ANTIGENS IN CELL MONOLAYERS
J.D. ORAM and A.J. CROOKS Microbiological Research Establishment, Porton Down, Salisbury, Wilts. SP4 0JG, U.K.
(Received 26 July 1978, accepted 8 August 1978)
A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-well polystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays using peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than the direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods.
INTRODUCTION L a b o r a t o r y m e t h o d s f o r t h e r a p i d diagnosis o f virus i n f e c t i o n s f r e q u e n t l y involve t h e e x a m i n a t i o n o f b i o p s y s p e c i m e n s f o r t h e p r e s e n c e o f virusspecific antigens in i n f e c t e d cells b y t h e use o f radio- or f l u o r e s c e n t - l a b e l l e d a n t i b o d i e s or, m o r e r e c e n t l y , e n z y m e - l i n k e d a n t i b o d i e s . Successful diagnosis d e p e n d s u p o n t h e p r e s e n c e o f c o m p a r a t i v e l y large a m o u n t s o f virus-specific antigens at a relatively late stage o f virus i n f e c t i o n . M u c h less a t t e n t i o n has b e e n given t o t h e d e t e c t i o n o f t h e l o w c o n c e n t r a t i o n s o f viruses w h i c h a r e p r e s e n t d u r i n g early stages o f i n f e c t i o n , in air o r w a t e r samples, or in t h e b l o o d used f o r t r a n s f u s i o n p u r p o s e s . D u e to its low antigenic mass, each virus particle b i n d s v e r y f e w a n t i b o d y m o l e c u l e s so t h a t t h e d i r e c t d e t e c t i o n o f l o w c o n c e n t r a t i o n s o f viruses is d i f f i c u l t o r i m p o s s i b l e . Using 12SI-labelled i m m u n o p u r i f i e d a n t i b o d y , S t r a n g e a n d Martin ( 1 9 7 2 ) f o u n d t h a t t h e l o w e r limit f o r t h e d i r e c t d e t e c t i o n o f c o l i p h a g e T7 was a b o u t 5 × l 0 s virions. A l m e i d a and W a t e r s o n ( 1 9 6 9 ) f o u n d , b y ' i m m u n e ' e l e c t r o n m i c r o s c o p y , a d i r e c t d e t e c t i o n l i m i t o f a b o u t 106 virions f o r s o m e a n i m a l viruses. H o w e v e r , l o w e r c o n c e n t r a t i o n s o f virus m a y b e d e t e c t e d if t h e mass o f virus antigens is a m p l i f i e d b y g r o w i n g t h e virus in a s u s c e p t i b l e h o s t cell. A n u m b e r o f labelled a n t i b o d y m e t h o d s h a v e b e e n used t o d e t e c t virus antigens in h o s t cells; t h u s F L A was used b y W h e e l o c k and T a m m ( 1 9 6 1 ) , R L A b y H a y a s h i et al. {1972), E L A b y A b e l s o n et al. {1969) and Wicker and Avra-
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meas (1969) and PAP by Dougherty et al. (1974). Although high multiplicities of infection were used in these and most subsequent studies, Carter (1969) reported the detection by the FLA method, of 100 plaque-forming units (PFU) of Semliki Forest virus (SFV) after cultivation in BHK cells for 18 h. Levitt et al. (1976) found a similar sensitivity for an indirect RLA assay used to detect Western Equine encephalomyelitis virus antigens in duck embryo cells. Despite the increasing use of host cells to produce detectable amounts of virus antigens, the quantitative aspects of the detection methods have not been fully investigated. In this investigation we have used a number of labelled antibody methods to detect virus antigens produced by SFV in primary chick embryo cells (CEC). We have attempted to define the optimal reaction conditions and have compared the sensitivities of different methods. METHODS
Virus
The A774 strain of SFV (Bradish et al., 1971) was passed once in CEC and 0.5 ml portions containing l 0 s PFU/ml were stored at --80°C; these were thawed just before use. Virus infectivity was titrated as PFU/ml in CEC suspended in agar (Bradish et al., 1971).
Preparation of antisera H y p e r i m m u n e sera against viruses were prepared in New Zealand White rabbits. For SFV, Venezuelan Equine encephalomyelitis virus (VEEV), strain TC83, and Langat virus, strain TP21, the rabbits received two intravenous injections, given 1 m o n t h apart, of 106 PFU. For influenza virus, t y p e A/WSN (HON1), several subcutaneous injections were made of about 1 mg (approximately 3 X 10 '2 virions) of purified virus emulsified in Freund's complete adjuvant. Sheep antibody to rabbit IgG fraction (SAR) was prepared b y the intramuscular injection of a sheep with 10 mg of normal rabbit IgG fraction emulsified in Freund's complete adjuvant. This was followed, at intervals of 8 weeks, by booster injections of 40 mg of IgG dissolved in saline.
Purification of antibodies The IgG fraction was prepared from normal or hyper-immune rabbit sera by the method of Sober et al. (1956). A n t i b o d y to SFV was immunopurified by the m e t h o d of Fitzgeorge and Bradish (1973). Anti-virus antibodies were titrated b y either the ring inhibition test described b y Fitzgeorge and Bradish (1973) or the haemagglutination inhibition (HI) test of Clarke and Casals (1958). SAR was purified b y adsorbing immune serum with Sepharose 4B gel coupled with a b o u t 2 mg/ml of rabbit IgG fraction. Serum (25 ml) was gently stirred with Sepharose-IgG gel (5 ml) at 37°C for 1 h and then at 4°C
299 for 16 h. The gel was washed on a Buchner funnel with phosphate-buffered saline, pH 7.4 (PBS) at 4°C until the filtrate was free of material absorbing at 280 nm and then extracted with 10 ml of 1% NaC1 + 0.1 M HC1 buffer, pH 1.2, at 0°C for 15 min. The extract was neutralized, centrifuged at 2 0 0 0 X g for 1 5 m i n to remove insoluble material and concentrated by vacuum dialysis to a b o u t 1 ml. All antibody preparations were stored, in 0.5 ml or 1 ml lots, at --20°C. Anti-virus antibody preparations were adsorbed 3 times with washed, methanol-fixed monolayers of CEC in 5 cm plastic Petri dishes. Antibody was diluted, normally 1/10, in a 2% solution of bovine gamma-globulin (BGG, fraction II, Armour Pharmaceutical Co Ltd., Eastbourne, Sussex) in PBS and 1 ml portions added to each dish which was mixed occasionally at room temperature for 2 h. Fab fragments were prepared by digestion of antibodies with papain as described by Porter (1959). The digests were dialyzed against water, with daily changes, at 4°C for 3--4 days. After centrifuging at 2000 X g for 30 min to remove the insoluble F c fragments, the Fab preparation was concentrated to about 5 mg/ml by vacuum dialysis at 0°C.
Radioactive labelling of antibody Anti-virus IgG fraction, Fab fragments and SAR were labelled with 12sI by the chloramine T method as described by Strange et al. (1971) except that each milligramme of antibody was reacted with between 2 and 4 mCi o f isotope. Preparations of 12SI-labelled antibody contained between 0.03 and 0.10 atoms of ~25I/molecule. Anti-virus IgG fraction and SAR were labelled with tritium by reaction with 3,5-3H-l-fluoro,2,4-dinitrobenzene ([3H]FDNB; 14.5 or 30 Ci/mmole) as described by Benbough and Martin (1976) except that each milligramme of antibody was reacted with from 2.5 to 5 mCi of [3H]FDNB. Preparations of [3H]anti-virus IgG fraction contained between 2.7 and 4.4 atoms of tritium/molecule and [3H]SAR contained between 10 and 35 atoms of tritium/molecule.
Conjugation of antibody with peroxidase Anti-virus IgG fraction and SAR were conjugated with horseradish peroxidase (HRP) by the m e t h o d of Nakane and Kawaoi (1974). The enzyme used was Sigma t y p e VI (Sigma London Chemical Co. Ltd., Poole, Dorset) with Rz values between 2.8 and 3.2. Conjugates were purified b y filtration through a column (96 mm X 1.6 cm) of Ultragel AcA-44 (LKB Instruments Ltd., South Croydon, Surrey), equilibrated with PBS at 4°C, with a flow rate o f 20 ml/h. The column was calibrated with proteins of known mol. wt. and fractions corresponding to a 1 + 1 conjugate of HRP and IgG (mol. wt. about 190,000) were pooled and stored, at a concentration of about 100 ~g/ ml, in 1 ml lots at --20°C.
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Peroxidase-antiperoxidase complexes (PAP) A preparation was kindly provided by Dr. R.E. Strange of this establishment. It was prepared as described by Sternberger (1974) and contained 3 mg/ml of protein and had a peroxidase : antiperoxidase mole ratio of 1.42 : 1.
Fluorescent labelling of antibody SAI~ was reacted with powdered fluorescein iso-thiocyanate (FITC, isomer 1, B.D.H., Poole, Dorset) as described by Goldman (1968) and untreated FITC removed by filtering the reaction mixture through a column (20 cm × 0.9 cm) of Sephadex G-50 equilibrated with PBS. Fluorescein-SAR (F-SAR) conjugates contained about 3 mg/ml of protein and had fluorescein : protein mole ratios of between 2.1 : 1 and 3.5 : 1. For use it was diluted 1/100 in a 0.25% solution of Evans blue stain in a mixture of foetal calf serum plus PBS (1 + 1).
Detection of virus antigens in cell monolayers Monolayers of CEC were prepared in 96-well polystyrene tissue culture plates (Microtest II, Cat. No. 3040, from Falcon, Oxnard, CA, U.S.A.). Suspensions of CEC in medium (medium 199 plus 10% foetal calf serum and 0.088% NaHCO3) were adjusted to about 106 cells/ml and 0.1 ml added to each well, the plates covered with Microtest film (Falcon) and incubated overnight at 37°C in an atmosphere of 5% CO2 plus 95% air. The confluent monolayers were infected with 0.1 ml of dilutions of virus or mock-infected with medium and incubated for various times at 37°C. The cultures were washed with a 1% solution in PBS of bovine plasma albumin (fraction V, Armour Pharmaceutical Co. Ltd., Eastbourne, Sussex), followed by PBS and then fixed with methanol as described by Robb (1973), except that the methanol was precooled to --20°C. Finally, the fixed cultures were dried in a stream of air. Before reaction with antibody, the fixed cultures were rinsed with a 2% solution of BGG in PBS. Antibodies were diluted in 2% BGG solution and filtered, just before use, through a Millipore filter (0.45 t~m) and 25 t~l portions added from a Pasteur pipette to each culture. After incubation at room temperature, usually for 2 h, excess antibody was shaken from the plate which was then washed 4 or 5 times with a 1% solution in PBS of Brij 35 (polyoxyethylene glycol, B.D.H., Poole, Dorset). Cultures reacted with RLA were extracted with 0.1 ml of 0.1% Brij 35 plus 0.2 M acetic acid for 30 min at room temperature (K.L. Martin, personal communication). The radioactivity of samples containing 125I-labelled antibodies was measured in a well-type sodium iodide crystal scintillation counter (automatic spectrometer, NE8311, Nuclear Enterprises, Edinburgh) with a counting efficiency of about 40%. Samples containing tritiated antibody were mixed with 20 vol of Bray's scintillation fluid and the radioactivity measured in a Philips liquid scintillation counter (model PW 4510; N.W.
301
Philips Gloeilampenfabrieken, Eindhoven, The Netherlands), with a counting efficiency for tritium of about 40%. Cultures reacted with ELA or PAP were incubated with 200 ~l of peroxidase reagent and examined after 1 h at room temperature. The reagent was a solution of 80 mg of 5-aminosalicyclic acid in 100 ml of 0.05 M sodium acetate buffer, p H 6 . 0 ; after decolorizing with active charcoal, 0.1 vol of 30% (w/v) H202 were added just before use. Normally, the plates were examined by eye although in a few experiments the contents of the wells were transferred to a spectrophotometer and measured at 450 nm. Both methods gave very similar results. Cultures stained with FLA were wetted with 1 drop of a mixture of glycerol plus PBS (1 + 1) and the inverted plates examined with a Vickers M41 Photoplan fluorescence microscope (Vickers Ltd., York). Incident lighting was used from an Osram HBO 200 mercury vapour lamp with a BG 12/2 mm exciter filter and a combination of filters (OG 1/1.5 mm and GG 9/1.5 mm) as a barrier filter. The cultures were scanned, using 10 X oculars and a 10 X objective, and those showing specifically fluorescing multicellular loci scored as virus-positive. RESULTS
Direct assays using RIA The o p t i m u m reaction conditions for the detection of SFV in monolayer cultures of CEC were investigated using 12SI-labelled preparations of immunopurified antibody, the IgG fraction from antisera and Fab fragments derived from the IgG fraction. Since the different antibody preparations gave very similar results, only those obtained with the IgG fraction are presented in detail. For convenience, some results are expressed as virus/control signal ratios, defined as: mean counts/min from infected cultures/mean counts/min from mock-infected cultures. The o p t i m u m time for the reaction of infected monolayers with labelled antibody was about 2 h (Fig. 1), longer reaction times resulted in higher backgrounds and lower virus/control signal ratios. A reaction time of 2 h was used in all subsequent work with both direct and indirect antibody methods. The a m o u n t of radioactivity bound by infected cultures were directly proportional to the concentration of labelled antibody (Fig. 2). Labelled antib o d y was normally used at a concentration of about 10 ~g/ml (HI titre of about 2). This gave relatively high signals and a reasonable e c o n o m y in labelled antibody. The counts from mock-infected cultures were only 2--3 times higher than the instrument background; this had the important practical advantage that signals from virus antigens could be recognised with some confidence. A threshold signal was established, for each Microtest plate, above which individual cultures were regarded as virus-positive but which excluded virtually all the mock-infected cultures. Thresholds based on the mean plus either 2 or 3 standard deviations of mock-infected cultures
302
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Fig. 1. Rate of uptake of 12SI-labelled anti-SFV IgG fraction by monolayers of CEC. Washed and fixed cultures (n = 8) were reacted with 12SI-labelled anti-SFV IgG (128 pg/ ml, 3.9 × 107 counts/min/ml) for the times shown. ©, mean counts/min from cultures infected with 100 PFU of SFV and incubated for 18 h; e, mean counts/min from mockinfected cultures; bars indicate standard deviations.
excluded about 95% or 100%, respectively, of the mock-infected cultures. Our results are based on a threshold of the mean plus 3 standard deviations. The sensitivity of the m e t h o d was determined after various times of virus replication. Results are expressed as detection limits, defined as the infectivity of the input virus (PFU/culture) required to produce virus-positive signals in 50% of the cultures. Detection limits were obtained by plotting the percentage of virus-positive cultures versus the infectivity of the inoculum for each incubation time. This gave approximately parallel plots (Fig. 3), with good agreement between replicates on different days. A plot of the derived detection limits versus time of incubation (Fig. 4) shows that the m e t h o d was relatively insensitive after short incubation periods, but was very sensitive after overnight replication of virus. Thus, after 8 h incubation, the detection limit was about 700 PFU/culture, or 7 × 103 PFU/ml in the inoculum, but increasing the incubation time of the cultures to 16 h resulted in a 100fold increase in sensitivity, with a detection limit of about 8 PFU/culture. Results obtained using all-labelled anti-SFV IgG fraction were very similar to those given by 12SI-labelled antibody except that lower radioactive signals were obtained, even though the radioactivities (counts/min/pg of protein) of the two preparations were similar. Since the signals from mock-infected cultures were n o t significantly higher than the instrument background, nonspecific adsorption of antibody was negligible. The sensitivity of the m e t h o d
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Fig. 2. Titration of 12SI-labelled anti-SFV IgG fraction in direct assays using RLA. Washed and fixed cultures (n = 8) were reacted with t2SI-labelled anti-SFV IgG at the dilutions shown (undiluted concentration, 250 pg/ml, 1.9 × l0 s counts/min/ml); ©, e, as in Fig. 1;~, virus/control signal ratios; bars indicate standard deviations.
(Fig. 4) was equal to that obtained using 12SI-labelled antibody except that no virus was detected at 8 h (highest inoculum: 2 0 0 0 PFU/culture). Hence, for all but the shortest periods o f virus replication, 3H-labelled antibody, which stored well at --20°C, was as effective as the 12SI-labelled preparations.
Direct assays using ELA The o p t i m u m conditions for the use o f anti-virus IgG-HRP conjugate were similar to those determined for direct RLA, except that high concentrations o f conjugate (above 50 gg/ml) gave false-positive reactions, presumably due to non-specific adsorption o f antibody. D e t e c t i o n limits obtained using a conjugate concentration o f 30 ~g/ml are given in Fig. 4. Similar values were obtained, using several batches o f conjugate. The m e t h o d was about half as sensitive as the direct RLA m e t h o d for the detection o f virus antigens in CEC.
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Fig. 3. Detection of SFV antigens in CEC by direct assays using RLA. CEC monolayers (n = 20) were infected with SFV and incubated for the following times; o, 8 h; ~, 12 h; D, 16 h; v, 18 h; e, 24 h; A, 40 h. Washed and fixed cultures were reacted with 12sIlabelled anti-SFV IgG, 2.5 pg/ml, 1.9 X 106 counts/min/ml. Cultures giving radioactive signals higher than the mean plus 3 standard deviations of the counts from mock-infected cultures (n = 16) were scored as virus-positive.
Detection o f virus antigens by indirect assays T h e m a i n a d v a n t a g e o f i n d i r e c t a n t i b o d y m e t h o d s is t h a t a single t y p e o f l a b e l l e d a n t i b o d y ( a n t i - I g G ) c a n be u s e d t o d e t e c t a v a r i e t y o f a n t i g e n s . S i n c e it is f r e q u e n t l y a s s e r t e d t h a t i n d i r e c t a n t i b o d y m e t h o d s are m o r e s e n s i t i v e t h a n d i r e c t p r o c e d u r e s , we h a v e c o m p a r e d t h e s e n s i t i v i t i e s o f a n u m b e r o f i n d i r e c t assays w i t h t h o s e o f t h e d i r e c t m e t h o d s .
Indirect assays using 3H-labelled S A R S i n c e p r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t t h e b a c k g r o u n d signals w e r e m u c h h i g h e r t h a n t h o s e o b t a i n e d b y d i r e c t R L A m e t h o d s , we i n v e s t i g a t e d several
305
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i 24
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Fig. 4. Detection limits for SFV by labelled antibody methods. Each point, derived from curves of the type shown in Fig. 3, represents the infectivity of the input virus required to give virus-positive signals in 50% of the cultures, o, 12SI-labelled anti-SFV IgG (n = 20); e, 3H-labelled anti-SFV IgG (n --- 20); ~, HRP-labelled anti-SFV IgG (n = 24); A, anti-SFV IgG followed by HRP-labelled SAR (n = 24); El, anti-SFV IgG followed by 3H-labelled SAR (n = 60); I , PAP (n = 12); ~, anti-SFV IgG followed by fluorescein-labelled SAR (n = 60).
reaction parameters in an attempt to minimise non-specific adsorption of antibody. Immunopurified anti-virus antibody, the IgG fraction from anti-virus antiserum and Fab fragments derived from the IgG fraction were each adsorbed 3 times with CEC and tested, at approximately equal concentrations, with cultures incubated with or without 100 PFU of SFV for 18 h. As expected, immunopurified antibody gave the best results with virus/control signal ratios, in replicate experiments, of 12.2 and 15.4. The IgG fraction gave virus/control signal ratios of 7.8 and 10.1 and unfractionated antiserum gave ratios of 3.9 and 4.1. Although Fab fragments gave low background counts, the signals from infected cultures were much lower than those given b y undigested antibody molecules (virus/control signal ratios 1.7 and 2.3), indicating that the F c portion of the anti-virus antibody molecule was important for the binding of [3H] SAR. Whereas in the direct RLA m e t h o d the virus/control signal ratios were directly related to antibody concentration, the use of high concentrations of
306
anti-virus antibody in the indirect method resulted in very high non-specific adsorption of antibody and low virus/control signal ratios (Fig. 5). In some experiments virus/control signal ratios of about 1 were obtained at high concentrations of antibody (1--2 mg/ml). The highest ratios were obtained using anti-SFV IgG concentrations of between 10 and 20 ~g/ml (HI titres of about 2--4). In most subsequent experiments anti-virus antibody preparations were used at an HI titre of about 2. High concentrations of immunopurified antivirus antibody also gave reduced virus/control signal ratios, although the effect was less marked than with IgG preparations. The effect of [3H] SAR concentration was determined with cultures which had been reacted with anti-virus IgG fraction at a concentration of 20 ~g/ml (HI titre -- 4). The highest virus/control signal ratio was obtained with a [3H]SAR concentration of about 10 ~g/ml; concentrations of [3H]SAR below about 5 gg/ml resulted in lower sensitivities. The sensitivity of the indirect RLA method using [3H]SAR was 2--5-fold
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Fig. 5. T i t r a t i o n o f a n t i - S F V IgG fraction in indirect assays using R L A . Washed and f i x e d cultures were reacted w i t h a n t i - S F V IgG at the d i l u t i o n s s h o w n ( u n d i l u t e d c o n c e n t r a t i o n , 1 5 . 2 m g / m l , HI titre, 8 0 0 0 ) , f o l l o w e d b y [ 3 H ] S A R at 1 3 . 5 pg/ml, 2.3 × 10 ? c o u n t s / r a i n / ml. o, m e a n o f c o u n t s / m i n f r o m cultures (n = 8) i n f e c t e d w i t h 1 0 0 P F U and i n c u b a t e d 18 h; bars indicate standard deviations; o, m e a n o f c o u n t s / m i n from m o c k - i n f e c t e d cultures (n = 8); A, virus/control signal ratios.
307 lower than that obtained using the direct R L A methods (Fig. 4). For example, the detection limits after cultivation of SFV for 16 h were a b o u t 8 and 40 PFU/culture for the direct and indirect R L A methods, respectively. The lower sensitivity of the indirect method appeared to be due partly to higher non-specific adsorption of antibody. At relatively short incubation times (8--12 h) there was a good correlation between the infectivity of the inoculum and the radioactive signal. However, no correlation was found after longer incubation periods, presumably since these allowed sufficient time for the synthesis of approximately equal amounts of virus antigen in all infected cultures. The specificity of the m e t h o d was examined by reacting SFV-infected cultures with normal rabbit serum, antibody to SFV or hyperimmune sera to the serologically unrelated VEEV, Langat virus or influenza virus. Only the anti-SFV antibody produced virus-positive signals. Cultures infected with VEEV, Langat virus or influenza virus and reacted with anti-SFV antibody followed by [ 3H] SAR gave virus-negative signals. The validity of results obtained b y the indirect assay was tested b y relating the radioactive signals obtained from individual cultures to the infectivities of the corresponding culture supernatant fluids. Cultures were infected with between 1.7 and 150 P F U and after incubation for 40 h the supernatant fluids were removed for infectivity titrations and the monolayers reacted with anti-virus antibody followed by [3H]SAR. Virus infectivity was detected in 39 of the 60 cultures tested, with virus yields between 3 × 104 and 1.4 X l 0 s PFU/culture (assay limit, 10 PFU/culture). With the exception of the two lowest-yielding cultures (3.4 X 104 PFU and 5 X 104 PFU) all the virus-producing cultures gave radioactive signals (range, 6529-17,179 counts/min) well above those (mean = 3200 counts/min; S.D. = 592) obtained from the 32 uninfected cultures. None of the virus-negative cultures gave a signal higher than the control values. Indirect assay using 1~SI-labelled S A R The o p t i m u m concentrations of anti-SFC antibody and ~25I-labelled SAR were very similar to those determined using [3H]SAR. The method was a b o u t as sensitive as the direct R L A method and rather more sensitive than the indirect R L A m e t h o d using [3H]SAR, possibly because the 12SI-label had a higher specific activity. Indirect assay using E L A The best discrimination between virus-infected and mock-infected cultures, and hence the highest sensitivity, was obtained using a b o u t 5 pg/ml of anti-virus IgG fraction followed by 5--10 pg of SAR-HRP conjugate, each for 2 h at room temperature. In a limited study, similar results were obtained using twice the concentrations of antibody and conjugate for a 1 h reaction time. The sensitivity of the m e t h o d was a b o u t the same as the direct ELA
308 m e t h o d and slightly higher than the indirect RLA m e t h o d using [3H]SAR (Fig. 4). Indirect assay using PAP As with the ELA m e t h o d , close attention to reaction conditions was essential, otherwise the mock-infected cultures gave non-specific colour reactions. Best results were obtained using anti-virus IgG fraction at a concentration of about 5 pg/ml followed by SAR at about 10 ~g/ml and then PAP at about 3 pg/ml, each for 2 h at room temperature. The m e t h o d was approximately twice as sensitive as the ELA m e t h o d and about as sensitive as the direct RLA m e t h o d (Fig. 4). Indirect assays using F L A The o p t i m u m reaction conditions were similar to those determined for other indirect methods. Immunopurified anti-virus antibody or anti-virus IgG fraction were used at concentrations of 5--10 gg/ml followed by F-SAR at about 30 t~g/ml. The detection limits (Fig. 4) were very similar to those obtained using [3H]SAR. Although Carter (1969) observed non-specific fluorescence in CEC, this was not a problem in this investigation in which we adsorbed the antibody with CEC and used Evans blue as a counterstain. DISCUSSION The sensitivity of a m e t h o d for the detection of virus antigens in monolayers of host cells depends on a number of factors including the efficiency of uptake of virus, the rate of virus replication and the efficiency of the detection system. Clearly, the efficiency of virus uptake and rate of virus replication is influenced by the choice of host cell and the cultural conditions. The efficiency of the detection system depends on the purity and avidity of antibody preparations, the extent of labelling of the antibody and the sensitivity of the m e t h o d used to detect the label. The effect of antibody purity was examined by the indirect RLA method. The virus/control signal ratios obtained using IgG preparations were about twice those given by unfractionated antiserum and there appeared to be little further advantage in using immunopurified antibody, the preparation of which requires relatively large amounts of antigen for use as an immunoadsorbent. We found that the sensitivities of some methods, especially indirect assays with RLA and ELA, were to some extent limited by the level of the background signals from uninfected cultures. Non-specific binding of anti-virus antibody appeared to be the most important factor and was considerably reduced by multiple adsorptions of antibody with host cells. It was further reduced by careful selection of the concentration of anti-virus antibody, previously found by Schieble and Cottam (1977) to be important in the detection of influenza virus antigens by the indirect RLA method. The back-
309 ground signals were also found to depend on the concentration of the second antibody (3H- or 125I-labelled SAR), with an optimum concentration (40 pg/ ml; 250 ng/well) very similar to that reported by Hutchinson and Ziegler (1974). However, using optimum reaction conditions for the indirect R L A method, the background signals were still 2--3-fold higher than those obtained in the direct method, which was more rapid to perform. In the direct R L A method, the highest virus/control signal ratios were obtained at relatively high concentrations of anti-virus antibody. These results appear to contradict the predictions of Kalmakoff et al. (1977) that the highest sensitivity is obtained using minimal amounts of labelled antibody. We confirm that the percentage of bound antibody falls with increase in labelled antibody concentration, although in our experiments the fall was small, i.e. from 0.25% at 5.9 ng/well to 0.14% at 740 ng/well. However, the most important factor, given low background signals, would appear to be the total amount, rather than percentage, of b o u n d antibody and we found that this was 40-fold higher, with only a 5-fold increase in the background signal, at the higher concentration of labelled antibody. We have used the m e t h o d of Benbough and Martin (1976) to tritiate antibodies and confirm that [3H]FDNB is a very effective labelling reagent and that tritiated antibodies can be stored for months at --20°C, without loss of serological reactivity. Although [3H]FDNB is a relatively expensive reagent, its use eliminates the need to radio-iodinate antibody at frequent intervals, with the attendant radiochemical hazard to health. Although the ELA m e t h o d has been used to detect or locate a variety of viruses in infected cells, most investigators have used high multiplicities of virus. We have found that low concentrations of virus can be detected by this method, provided that the reaction conditions were carefully controlled. The main disadvantage of both ELA and PAP methods was that antibodies and conjugates appeared to bind, non-specifically, to host cells so that background readings were much higher than in sandwich-antibody assays. Although cultures producing high yields of virus gave unambiguous results, it was more difficult to discriminate between low-yielding and uninfected cultures. Nevertheless, direct and indirect assays using ELA proved to be reliable and sensitive. The direct assay using ELA was especially easy to perform and was the most rapid of the methods examined. Although the PAP method was very sensitive, the assays were relatively slow and tedious to perform. We found it difficult to complete the PAP assays within a normal working day, although the reaction times could probably be shortened with little loss in sensitivity. Microplate assays with ELA are well suited to automation (Ruitenberg et al., 1976) and to full containment for use with dangerous pathogens. Results obtained b y each method were reproducible and consistent between batches of virus or labelled antibody. We conclude that the best method, of those which we have examined, for the detection of a single or limited number of types of virus would be direct assays using ELA or
310 R L A , using tritiated a n t i b o d y . Where an indirect m e t h o d is preferred, indirect assays using E L A a p p e a r e d t o be slightly m o r e sensitive and easier to p e r f o r m t h a n t h e o t h e r indirect m e t h o d s . ACKNOWLEDGEMENTS We wish to t h a n k C.J. Bradish and R.B. F i t z g e o r g e f o r supplies o f virus and antisera, R.E. Strange for p r o v i d i n g a sample o f PAP and these and o t h e r colleagues f o r m u c h advice and c o m m e n t . REFERENCES Abelson, H.T., G.H. Smith, H.A. Hoffmann and W.P. Rowe, 1969, J. Natl. Cancer Inst. 42,497. Almeida, J.D. and A.P. Waterson, 1969, Adv. Virus Res. 15,307. Benbough, J.E. and K.L. Martin, 1976, J. Appl. Bacteriol. 41, 47. Bradish, C.J., K. Allner and H.B. Maber, 1971, J. Gen. Virol. 12,141. Carter, G.B., 1969, J. Gen. Virol. 4,139. Clarke, D.H. and J. Casals, 1958, Am. J. Trop. Med. Hyg. 7,561. Dougherty, R.M., H.S. DiStefano and A.A. Marucci, 1974, in: Viral Immunodiagnosis, eds. E. Kurstak and R. Morisett (Academic Press, New York) p. 89. Fitzgeorge, R.B. and C.J. Bradish, 1973, Immunochemistry 10, 21. Goldman, M., 1968, Fluorescent Antibody Methods (Academic Press, New York) p. 101. Hayashi, K.J., J. Rosenthal and H.L. Notkins, 1972, Science 176,516. Hutchinson, H.D. and D.W. Ziegler, 1974, Appl. Microbiol. 28,935. Kalmakoff, J., A.J. Parkinson, A.M. Crawford and B.R.G. Williams, 1977, J. Immunol. Methods 14, 73. Levitt, N.H., H.V. Miller and G.A. Eddy, 1976, J. Clin. Microbiol. 4, 382. Nakane, P.K. and A. Kawaoi, 1974, J. Histochem. Cytochem. 22, 1084. Porter, R.R., 1959, Biochem. J. 73,119. Robb, J.A., 1973, in: Tissue Culture Methods and Application, eds. P.F. Kruse and M.K. Patterson (Academic Press, New York) p. 517. Ruitenberg, E.J., B.M.J. Brosi and P.A. Steerenberg, 1976, J. Clin. Microbiol. 3,541. Schieble, J.H. and D. Cottam, 1977, Infect. Immun. 15, 66. Sober, H.A., F.J. Gutter, M.M. Wyckoff and E.A. Peterson, 1956, J. Am. Chem. Soc. 78,756. Sternberger, L.A., 1974, Immunocytochemistry (Prentice-Hall, Englewood Cliffs, NJ) p. 219. Strange, R.E. and K.L. Martin, 1972, J. Gen. Microbiol. 72,127. Strange, R.E., E.O. Powell and T.W. Pearce, 1971, J. Gen. Microbiol. 67,349. Wheelock, E.F. and I. Tamm, 1961, J. Exp. Med. 113,301. Wicker, R. and S. Avrameas, 1969, J. Gen. Virol. 4,465.
