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A microwell immunoperoxidase test for screening hybridomas and for diagnosing newcastle disease virus and sendai virus
Journal oflmmunologicaIMethods, 61 (1983) 165-170 Elsevier
165
A Microwell Immunoperoxidase Test for Screening Hybridomas and for Diagnosing Newcastle Disease Virus and Sendai Virus P.H. Russell, P.C. Griffiths and M.J. Cannon Department of Microbiology' and Parasitology', Royal Veterinary College (University of London), Royal College Street, London N W 1 0 T U , U,K.
(Received 10 September 1982, accepted 10 January 1983)
A cell counting assay was developed to measure the infectivity of Sendai virus and Newcastle disease virus by indirect immunoperoxidase. It was used to monitor monoclonal antibodies against Newcastle disease virus, antibody to Sendai virus in mice and tracheal infectivity in chickens. Ke~,~,vords: indirect immunoperoxidase - - monoclonal antibodies - - viral diagnosis - - Newcastle disease virus - - Sendai virus
Microwell enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection and monoclonal antibody (MCA) screening. An ELISA for Newcastle disease virus (NDV) was found to be unsatisfactory (see below) and an indirect immunoperoxidase (IIP) test was developed in its place. Mice were immunised with allantoic-grown N D V and their sera, or hybridoma supernates, were tested on M a d i n - D a r b y bovine kidney (MDBK) cells infected with N D V as compared with Sendai virus. This circumvented the non-specific antibody forming cell responses to chicken antigen(s) or to neuraminidase-revealed interspecies antigen(s) as described by Russell (1981). The IIP test evolved from 2 assays: an IIP test for MCA to intermediate filaments or T antigen (Lane and Lane, 1981) and an SV infectivity assay which counted cells stained by indirect immunofluorescence (IIF) (Kashiwazaki et al., 1965). The I I F assay for SV was satisfactorily reproduced with N D V and then changed from I I F on glass slides with acetone fixation, to IIP on microwells with formalin fixation.
Materials and Methods
Microwells of M D B K cells (less that 200 passages) were infected overnight with dilutions of virus in 100 ~tl of RPMI. On the next day the cells were fixed with 200 0022-1759/83/$03.00 @ 1983 Elsevier Science Publishers B.V.
166
/zl of 10% formol-saline and then washed in phosphate-buffered saline-Dulbecco's A (PBSA). The antiviral serum was diluted 1:400-1000 in PBSA and added to the fixed cells in 50 /~l volumes for 45 min and then washed off and replaced by a 1 : 2 0 0 - 4 0 0 dilution of antiglobulin conjugated to horseradish peroxidase. At each wash the wells went through 3 washing cycles, being filled with 200 ~1 PBSA and then emptied by a flick and blot. Three antiglobulins were used: rabbit anti-mouse immunoglobulin (Ig) conjugate (Dako, Copenhagen); goat anti-rabbit lg conjugate (Miles-Yeda, Israel); sheep anti-chicken Ig (a gift from Dr. lvanyi, Wellcome Research Laboratories) conjugated in this laboratory by the method of Johnson et al. (1980). Antibody binding was visualised by treatment with PBSA containing 0.06% hydrogen peroxidase and 2% ethanol saturated with orthodianisidine (Lane and Lane, 1981). Infected cells showed brown particulate antigen in the cytoplasm and a brown cell membrane. Diffuse and pale cytoplasmic staining of all cells in the inner part of the well was sometimes apparent if 1% formalin was used instead of 10% formalin or if the substrate was left on for 60 instead of 15 rain.
Results
The liP test was first used as an infectivity assay (Table I). The number of cells containing antigen after overnight infection was proportional to the dilution of adsorbed virus, and the infectivity titre of the original inoculum was much the same whether counted with antisera from mice, rabbits or chickens (Table I). The titre of N D V was 106.9 107.6 IU per 0.1 ml compared with 10 s7 plaque forming units (PFU) per 1 ml and the titre of SV was 1 0 6 2 - 1 0 6 7 IU compared with 10 s° PFU. The titres of N D V and SV were therefore about 3-fold less by liP cell counting than by plaque assay. This was probably related to the less efficient adsorption in microwells with an average inoculum depth of 3.5 m m compared with 0.5 m m in the plaque assay. Adaptations of the l i p test for assaying infectivity were used to measure antibody binding and virus neutralization. In binding assays serum dilutions were added to fixed microwells containing 250 infected cells. In virus neutralization tests, 250 IU of virus and an equal volume of diluted serum were reacted in 100 t~l of RPMI for 2 h at 37°C in V-bottomed wells. The reaction then was assayed for residual IU (see above) with antiviral antibody from a second species. Results with ELISA, haemagglutination inhibition (HI), liP binding and neutralization tests were compared for specificity of antibody detection. HI, liP and neutralization but not ELISA results with antiviral sera showed orthogonal specificity between N D V and SV (Table II). Specific serum titres were highest by IIP (104-1045), intermediate by virus neutralization (1032-103.5 ) and as expected by H I (1027-1029). A possible artefact of the virus neutralization test with conventional antiserum to whole virus was that serum blocking could be scored as neutralization. This was checked in a pseudo-neutralization test when virus was adsorbed for 2 h at 37°C before adding serum overnight. Chicken or rabbit sera both specifically blocked sera from the other 2 species, i.e., anti-NDV serum from chickens blocked anti-NDV but
NDV
N DV NDV
NDV ND V NDV
SV
SV
1
2
3
4
5
rabbit
mouse
mouse chicken rabbit
chicken rabbit
mouse
Source of specific serum for l IP ~
6.2
4
6.69
6.2
6.88 7.02
4.3
6.74
6.23 c
7.33 6.83 7.03
4.6
6.78
6.0
7.25 6.86 7.02
7.17 b
4.9
6.65
7.35 6.99 7.06
7.14
5.2
6.69
7.42 7.0 7.07
7.2 7.56
7.19
5.5
6.65
7.45 6.89 7.1
7.18 7.53
7.13 ~
5.8
7.48 6.87 7.18
7.14 7.57
7.0 ~
6.1
7.04 7.57
6.4
Infectivity titre of original inoculum calculated from cells stained at these Iog~0 dilutions of stock virus:
a Sera were pooled from 2- 10 animals following these immunisation procedures: Mouse anti-NDV, 3 intraperitoneal inoculations of 30/~g of sucrose banded NDV. Chicken anti-NDV, 5 intramuscular inoculations of inactivated N D V (Ulster) vaccine (Newcadin, Evans, Liverpool). Rabbit anti-NDV, multiple intravenous inoculation of 15 # g of N D V by the protocol of Webster (1968). Mouse anti-SV, 2 intraperitoneal inoculations of 0.1 SV allantoic fluid containing 108.6 P F U / m l . Rabbit anti-SV, donated by Dr. A.F. Bradburne, Wellcome Research Labs. b Each figure represents log10 titre of virus in 0.1 ml inoculum. c Standard error of the mean titre was 2-10% for 4 or 6 replicate wells unless marked ~ when it was 12-30%.
Virus
Exp.
T H E M E A S U R E M E N T OF NDV A N D SV INF EC TIV ITY TITRES BY O V E R N I G H T I N F E C T I O N OF M I C R O W E L L S A N D C E L L C O U N T I N G BY liP
TABLE 1
168 TABLE I1 THE MEASUREMENT OF ANTIVIRAL ANTIBODY BY HI, liP AND VN Serum ~'
Logl0 titre of serum to NDV or SV ELISA b NDV
Mouse anti-NDV Mouse anti-SV Chicken anti-NDV Rabbit anti-NDV Rabbit anti-SV SPF mice (41) Conventionally housed mice (28)
4 5
HI c SV 3 3
NDV 2.7
liP SV <1 2.9 <1 <1 2.6 <1 1.9 + 1.1
NDV 4
VN SV
NDV
SV
<1 4
3.2 <1 3.9 3.5
<1 3 1.5 <1
1
4
3 4
~' Same serum pools as in Table I. SPF and conventional mice were tested individually and sample sizes are in parentheses. b ELISA, 10 ,ag of sucrose-banded virus was adsorbed overnight to microwells and the bovine serum albumin was used as a serum block at 2% and carrier at 0.2%. Positive binding was 2-fold above the antiglobulin conjugate's background, which was the same with or without normal serum. HI, sera were reacted with an equal volume of 8 HAU of allantoic virus in V-bottomed microwell plates and HI was read by streaming.
n o t a n t i - S V s e r u m f r o m r a b b i t s a n d m i c e . S u c h b l o c k i n g o c c u r r e d in d i l u t i o n s of 1 : 50 1 : 100 a n d h a d n o t b e e n s c o r e d as n e u t r a l i z a t i o n b e c a u s e n e u t r a l i z a t i o n t i t r e s w e r e at l e a s t 1 0 0 - f o l d h i g h e r . T h e l i P t e s t w a s u s e d to select M C A w h i c h b i n d a n d n e u t r a l i z e N D V , to d e t e c t s u b c l i n i c a l S V in m o u s e c o l o n i e s a n d t o i s o l a t e N D V f r o m c h i c k e n t r a c h e a . O n e t h o u s a n d s i n g l e c l o n e s w e r e s c r e e n e d b y I I P f o r M C A to N D V - i n f e c t e d cells. N i n e c l o n e s u p e r n a t e s t h a t b o u n d N D V b u t n o t S V w e r e f u r t h e r i n v e s t i g a t e d a n d 17 clones which bound both NDV and SV were discarded. The frequency of anti-NDV c l o n e s w a s a b o u t 1%. T h i s l o w f r e q u e n c y w a s n o t d u e to i n s e n s i t i v i t y o f t h e I I P test s i n c e m e d i u m f r o m g r o w i n g f l a s k c u l t u r e s h a d l i P t i t r e s o f 1015 104 ( r e s u l t s n o t shown). T h e l i P w a s a l s o u s e d to s e l e c t t h e 9 M C A c l o n e s b y v i r u s n e u t r a l i z a t i o n . B e c a u s e n e u t r a l i z a t i o n t i t r e s o f c o n v e n t i o n a l s e r a w e r e a b o u t 10-fold l o w e r t h a n I I P titres, t h e M C A s w e r e p a s s a g e d in m i c e . F o u r o f t h e 9 M C A a s c i t e s f l u i d s h a d n e u t r a l i z a tion titres of 102-10 s (results not detailed). These MCAs were amongst the 5 which s t a i n e d t h e cell m e m b r a n e b y l i P a n d t h e s u r f a c e o f l i v i n g P815 cells b y l I F . T h e o t h e r 4 M C A s s t a i n e d t h e c y t o p l a s m ( F i g . 1). S V is o f t e n e n d e m i c in a n i m a l h o u s e s ( G a n n o n a n d C a r t h e w , 1980) a n d m a y i n t e r f e r e w i t h c e l l - m e d i a t e d i m m u n e r e s p o n s e s , e.g., n a t u r a l killer cells ( C l a r k et al., 1979). W h e n s e r a w e r e s a m p l e d f r o m a g n o t o b i o t i c - d e r i v e d a n d c l o s e d b r e e d i n g c o l o n y of m i c e , n o a n t i b o d i e s to S V o r N D V w e r e d e t e c t e d b y l i P o r H I . W h e n s t o c k f r o m t h e s a m e c o l o n y w a s k e p t in a n o p e n a n i m a l h o u s e w h i c h c o n t a i n e d m i c e f r o m o t h e r c o l o n i e s t h e y h a d I I P a n d H I t i t r e s to S V b u t n o t N D V ( T a b l e I1). T h e
169
A
Fig. I. A: IIP staining of fixed NDV (Ulster)-infected MDBK cells to show discrete intracytoplasmic staining by MCA 479. B: IIP staining of fixed NDV (Ulster)-infected MDBK cells, to show predominant cell membrane staining by MCA 86. C: IIF staining of live NDV (Ulster)-infected P815 cells to show ring fluorescence of the external plasma membrane by MCA 86.
l i P test w a s a b e t t e r s e r o l o g i c a l test t h a n H I : s e r u m t i t r e s w e r e 10-fold h i g h e r , pretreatment of sera was not necessary and the infected microtitre plates could be s t o r e d at 5 ° C f o r s e v e r a l m o n t h s .
170
The IIP test was also used to isolate N D V in a pathogenesis experiment. Chicks were immunosuppressed with cyclophosphamide (CY) (Linna et al., 1972) and infected at 1 week old with 105 PFU of N D V (La Sota) as nasal droplets. Three days later 2 of 3 CY treated or control birds contained 10 3.9- 10 4.8 IU per trachea whereas 1 of 3 contained less than 10 ~3 IU. At 7 days 3 of 3 CY treated birds contained 105 2-1061 IU of NDV per trachea whereas 3 of 3 control birds contained less than 10 13 IU. Immunosuppression by CY had thereby enhanced the replication of NDV.
Discussion liP was therefore suited to routine assays for infectivity and antibody. The advantages of the IIP assay compared with plaque assays for measuring infectivity are: speed, i.e., overnight compared with 3 days; economy of materials 0.3 cm 2 compared with 20 cm 2 of cell monolayer and serological definition of the infectious agent. The IIP assay described has been used for an avirulent strain of NDV (Ulster), and for SV, both of which fail to spread between M D B K cells in the absence of trypsin and remain localised to the initially infected cells. When virulent strains of NDV, e.g., Essex '70, were use& it was possible to count foci of infected cells instead of individual cells. The use of the IIP test in serology has several advantages over ELISA and HI tests. It requires at least 100-fold less virus than HI or ELISA; it is a more sensitive measure of antibody than HI; it has better specificity than ELISA; and it gives a staining pattern which may be detailed and used to predict whether an MCA is likely to neutralize.
Acknowledgements This work was supported by a project grant from the Wellcome Trust. We thank Dr. Alexander of the Central Veterinary Laboratory (CVL) for encouragement and the CVL, Wellcome Research Laboratories and National Institute for Medical Research for reagents and cells.
References Clark. E.A., P.H. Russell, M. Egghart and M.A. Horton, 1979, lnt. J. Cancer 24, 688. Gannon, J. and P. Carthew, 1980, Lab. Anita. 14, 309. Johnson, R.B., R.M. Libby and R.M. Nakamura, 1980, J. Immunoassay 1, 27. Kashiwazaki, H., H. Homma and N. Ishida, 1965, Proc. Soc. Exp. Biol. Med. 120. 134. Lane, D.P. and E.B. Lane, 1981, J. Immunol. Methods 47, 303. Linna, I.J., D. Frommel and R.A. Good, 1972, Int. Arch. Allergy 42, 20. Russell, P.H., 1981, Immunology 44, 781. Webster, R.G., 1968, Immunology 14, 39.
165
A Microwell Immunoperoxidase Test for Screening Hybridomas and for Diagnosing Newcastle Disease Virus and Sendai Virus P.H. Russell, P.C. Griffiths and M.J. Cannon Department of Microbiology' and Parasitology', Royal Veterinary College (University of London), Royal College Street, London N W 1 0 T U , U,K.
(Received 10 September 1982, accepted 10 January 1983)
A cell counting assay was developed to measure the infectivity of Sendai virus and Newcastle disease virus by indirect immunoperoxidase. It was used to monitor monoclonal antibodies against Newcastle disease virus, antibody to Sendai virus in mice and tracheal infectivity in chickens. Ke~,~,vords: indirect immunoperoxidase - - monoclonal antibodies - - viral diagnosis - - Newcastle disease virus - - Sendai virus
Microwell enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection and monoclonal antibody (MCA) screening. An ELISA for Newcastle disease virus (NDV) was found to be unsatisfactory (see below) and an indirect immunoperoxidase (IIP) test was developed in its place. Mice were immunised with allantoic-grown N D V and their sera, or hybridoma supernates, were tested on M a d i n - D a r b y bovine kidney (MDBK) cells infected with N D V as compared with Sendai virus. This circumvented the non-specific antibody forming cell responses to chicken antigen(s) or to neuraminidase-revealed interspecies antigen(s) as described by Russell (1981). The IIP test evolved from 2 assays: an IIP test for MCA to intermediate filaments or T antigen (Lane and Lane, 1981) and an SV infectivity assay which counted cells stained by indirect immunofluorescence (IIF) (Kashiwazaki et al., 1965). The I I F assay for SV was satisfactorily reproduced with N D V and then changed from I I F on glass slides with acetone fixation, to IIP on microwells with formalin fixation.
Materials and Methods
Microwells of M D B K cells (less that 200 passages) were infected overnight with dilutions of virus in 100 ~tl of RPMI. On the next day the cells were fixed with 200 0022-1759/83/$03.00 @ 1983 Elsevier Science Publishers B.V.
166
/zl of 10% formol-saline and then washed in phosphate-buffered saline-Dulbecco's A (PBSA). The antiviral serum was diluted 1:400-1000 in PBSA and added to the fixed cells in 50 /~l volumes for 45 min and then washed off and replaced by a 1 : 2 0 0 - 4 0 0 dilution of antiglobulin conjugated to horseradish peroxidase. At each wash the wells went through 3 washing cycles, being filled with 200 ~1 PBSA and then emptied by a flick and blot. Three antiglobulins were used: rabbit anti-mouse immunoglobulin (Ig) conjugate (Dako, Copenhagen); goat anti-rabbit lg conjugate (Miles-Yeda, Israel); sheep anti-chicken Ig (a gift from Dr. lvanyi, Wellcome Research Laboratories) conjugated in this laboratory by the method of Johnson et al. (1980). Antibody binding was visualised by treatment with PBSA containing 0.06% hydrogen peroxidase and 2% ethanol saturated with orthodianisidine (Lane and Lane, 1981). Infected cells showed brown particulate antigen in the cytoplasm and a brown cell membrane. Diffuse and pale cytoplasmic staining of all cells in the inner part of the well was sometimes apparent if 1% formalin was used instead of 10% formalin or if the substrate was left on for 60 instead of 15 rain.
Results
The liP test was first used as an infectivity assay (Table I). The number of cells containing antigen after overnight infection was proportional to the dilution of adsorbed virus, and the infectivity titre of the original inoculum was much the same whether counted with antisera from mice, rabbits or chickens (Table I). The titre of N D V was 106.9 107.6 IU per 0.1 ml compared with 10 s7 plaque forming units (PFU) per 1 ml and the titre of SV was 1 0 6 2 - 1 0 6 7 IU compared with 10 s° PFU. The titres of N D V and SV were therefore about 3-fold less by liP cell counting than by plaque assay. This was probably related to the less efficient adsorption in microwells with an average inoculum depth of 3.5 m m compared with 0.5 m m in the plaque assay. Adaptations of the l i p test for assaying infectivity were used to measure antibody binding and virus neutralization. In binding assays serum dilutions were added to fixed microwells containing 250 infected cells. In virus neutralization tests, 250 IU of virus and an equal volume of diluted serum were reacted in 100 t~l of RPMI for 2 h at 37°C in V-bottomed wells. The reaction then was assayed for residual IU (see above) with antiviral antibody from a second species. Results with ELISA, haemagglutination inhibition (HI), liP binding and neutralization tests were compared for specificity of antibody detection. HI, liP and neutralization but not ELISA results with antiviral sera showed orthogonal specificity between N D V and SV (Table II). Specific serum titres were highest by IIP (104-1045), intermediate by virus neutralization (1032-103.5 ) and as expected by H I (1027-1029). A possible artefact of the virus neutralization test with conventional antiserum to whole virus was that serum blocking could be scored as neutralization. This was checked in a pseudo-neutralization test when virus was adsorbed for 2 h at 37°C before adding serum overnight. Chicken or rabbit sera both specifically blocked sera from the other 2 species, i.e., anti-NDV serum from chickens blocked anti-NDV but
NDV
N DV NDV
NDV ND V NDV
SV
SV
1
2
3
4
5
rabbit
mouse
mouse chicken rabbit
chicken rabbit
mouse
Source of specific serum for l IP ~
6.2
4
6.69
6.2
6.88 7.02
4.3
6.74
6.23 c
7.33 6.83 7.03
4.6
6.78
6.0
7.25 6.86 7.02
7.17 b
4.9
6.65
7.35 6.99 7.06
7.14
5.2
6.69
7.42 7.0 7.07
7.2 7.56
7.19
5.5
6.65
7.45 6.89 7.1
7.18 7.53
7.13 ~
5.8
7.48 6.87 7.18
7.14 7.57
7.0 ~
6.1
7.04 7.57
6.4
Infectivity titre of original inoculum calculated from cells stained at these Iog~0 dilutions of stock virus:
a Sera were pooled from 2- 10 animals following these immunisation procedures: Mouse anti-NDV, 3 intraperitoneal inoculations of 30/~g of sucrose banded NDV. Chicken anti-NDV, 5 intramuscular inoculations of inactivated N D V (Ulster) vaccine (Newcadin, Evans, Liverpool). Rabbit anti-NDV, multiple intravenous inoculation of 15 # g of N D V by the protocol of Webster (1968). Mouse anti-SV, 2 intraperitoneal inoculations of 0.1 SV allantoic fluid containing 108.6 P F U / m l . Rabbit anti-SV, donated by Dr. A.F. Bradburne, Wellcome Research Labs. b Each figure represents log10 titre of virus in 0.1 ml inoculum. c Standard error of the mean titre was 2-10% for 4 or 6 replicate wells unless marked ~ when it was 12-30%.
Virus
Exp.
T H E M E A S U R E M E N T OF NDV A N D SV INF EC TIV ITY TITRES BY O V E R N I G H T I N F E C T I O N OF M I C R O W E L L S A N D C E L L C O U N T I N G BY liP
TABLE 1
168 TABLE I1 THE MEASUREMENT OF ANTIVIRAL ANTIBODY BY HI, liP AND VN Serum ~'
Logl0 titre of serum to NDV or SV ELISA b NDV
Mouse anti-NDV Mouse anti-SV Chicken anti-NDV Rabbit anti-NDV Rabbit anti-SV SPF mice (41) Conventionally housed mice (28)
4 5
HI c SV 3 3
NDV 2.7
liP SV <1 2.9 <1 <1 2.6 <1 1.9 + 1.1
NDV 4
VN SV
NDV
SV
<1 4
3.2 <1 3.9 3.5
<1 3 1.5 <1
1
4
3 4
~' Same serum pools as in Table I. SPF and conventional mice were tested individually and sample sizes are in parentheses. b ELISA, 10 ,ag of sucrose-banded virus was adsorbed overnight to microwells and the bovine serum albumin was used as a serum block at 2% and carrier at 0.2%. Positive binding was 2-fold above the antiglobulin conjugate's background, which was the same with or without normal serum. HI, sera were reacted with an equal volume of 8 HAU of allantoic virus in V-bottomed microwell plates and HI was read by streaming.
n o t a n t i - S V s e r u m f r o m r a b b i t s a n d m i c e . S u c h b l o c k i n g o c c u r r e d in d i l u t i o n s of 1 : 50 1 : 100 a n d h a d n o t b e e n s c o r e d as n e u t r a l i z a t i o n b e c a u s e n e u t r a l i z a t i o n t i t r e s w e r e at l e a s t 1 0 0 - f o l d h i g h e r . T h e l i P t e s t w a s u s e d to select M C A w h i c h b i n d a n d n e u t r a l i z e N D V , to d e t e c t s u b c l i n i c a l S V in m o u s e c o l o n i e s a n d t o i s o l a t e N D V f r o m c h i c k e n t r a c h e a . O n e t h o u s a n d s i n g l e c l o n e s w e r e s c r e e n e d b y I I P f o r M C A to N D V - i n f e c t e d cells. N i n e c l o n e s u p e r n a t e s t h a t b o u n d N D V b u t n o t S V w e r e f u r t h e r i n v e s t i g a t e d a n d 17 clones which bound both NDV and SV were discarded. The frequency of anti-NDV c l o n e s w a s a b o u t 1%. T h i s l o w f r e q u e n c y w a s n o t d u e to i n s e n s i t i v i t y o f t h e I I P test s i n c e m e d i u m f r o m g r o w i n g f l a s k c u l t u r e s h a d l i P t i t r e s o f 1015 104 ( r e s u l t s n o t shown). T h e l i P w a s a l s o u s e d to s e l e c t t h e 9 M C A c l o n e s b y v i r u s n e u t r a l i z a t i o n . B e c a u s e n e u t r a l i z a t i o n t i t r e s o f c o n v e n t i o n a l s e r a w e r e a b o u t 10-fold l o w e r t h a n I I P titres, t h e M C A s w e r e p a s s a g e d in m i c e . F o u r o f t h e 9 M C A a s c i t e s f l u i d s h a d n e u t r a l i z a tion titres of 102-10 s (results not detailed). These MCAs were amongst the 5 which s t a i n e d t h e cell m e m b r a n e b y l i P a n d t h e s u r f a c e o f l i v i n g P815 cells b y l I F . T h e o t h e r 4 M C A s s t a i n e d t h e c y t o p l a s m ( F i g . 1). S V is o f t e n e n d e m i c in a n i m a l h o u s e s ( G a n n o n a n d C a r t h e w , 1980) a n d m a y i n t e r f e r e w i t h c e l l - m e d i a t e d i m m u n e r e s p o n s e s , e.g., n a t u r a l killer cells ( C l a r k et al., 1979). W h e n s e r a w e r e s a m p l e d f r o m a g n o t o b i o t i c - d e r i v e d a n d c l o s e d b r e e d i n g c o l o n y of m i c e , n o a n t i b o d i e s to S V o r N D V w e r e d e t e c t e d b y l i P o r H I . W h e n s t o c k f r o m t h e s a m e c o l o n y w a s k e p t in a n o p e n a n i m a l h o u s e w h i c h c o n t a i n e d m i c e f r o m o t h e r c o l o n i e s t h e y h a d I I P a n d H I t i t r e s to S V b u t n o t N D V ( T a b l e I1). T h e
169
A
Fig. I. A: IIP staining of fixed NDV (Ulster)-infected MDBK cells to show discrete intracytoplasmic staining by MCA 479. B: IIP staining of fixed NDV (Ulster)-infected MDBK cells, to show predominant cell membrane staining by MCA 86. C: IIF staining of live NDV (Ulster)-infected P815 cells to show ring fluorescence of the external plasma membrane by MCA 86.
l i P test w a s a b e t t e r s e r o l o g i c a l test t h a n H I : s e r u m t i t r e s w e r e 10-fold h i g h e r , pretreatment of sera was not necessary and the infected microtitre plates could be s t o r e d at 5 ° C f o r s e v e r a l m o n t h s .
170
The IIP test was also used to isolate N D V in a pathogenesis experiment. Chicks were immunosuppressed with cyclophosphamide (CY) (Linna et al., 1972) and infected at 1 week old with 105 PFU of N D V (La Sota) as nasal droplets. Three days later 2 of 3 CY treated or control birds contained 10 3.9- 10 4.8 IU per trachea whereas 1 of 3 contained less than 10 ~3 IU. At 7 days 3 of 3 CY treated birds contained 105 2-1061 IU of NDV per trachea whereas 3 of 3 control birds contained less than 10 13 IU. Immunosuppression by CY had thereby enhanced the replication of NDV.
Discussion liP was therefore suited to routine assays for infectivity and antibody. The advantages of the IIP assay compared with plaque assays for measuring infectivity are: speed, i.e., overnight compared with 3 days; economy of materials 0.3 cm 2 compared with 20 cm 2 of cell monolayer and serological definition of the infectious agent. The IIP assay described has been used for an avirulent strain of NDV (Ulster), and for SV, both of which fail to spread between M D B K cells in the absence of trypsin and remain localised to the initially infected cells. When virulent strains of NDV, e.g., Essex '70, were use& it was possible to count foci of infected cells instead of individual cells. The use of the IIP test in serology has several advantages over ELISA and HI tests. It requires at least 100-fold less virus than HI or ELISA; it is a more sensitive measure of antibody than HI; it has better specificity than ELISA; and it gives a staining pattern which may be detailed and used to predict whether an MCA is likely to neutralize.
Acknowledgements This work was supported by a project grant from the Wellcome Trust. We thank Dr. Alexander of the Central Veterinary Laboratory (CVL) for encouragement and the CVL, Wellcome Research Laboratories and National Institute for Medical Research for reagents and cells.
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