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Double-antibody sandwich ELISA for detection of infectious bursal disease virus
Br. vet. J. (1991) . 147, 25 1
DOUBLE-ANTIBODY SANDWICH ELISA FOR DETECTION OF INFECTIOUS BURSAL DISEASE VIRUS
AMIYA KUMAR* and A . T. RAOt Department of Pathology, Orissa Veterinary College, Bhubaneswar-751003, India SUMMARY A double-antibody sandwich ELISA was employed for detection of IBD virus in bursal suspension . Fifty IBD-free white leghorn chickens aged 5 weeks were experimentally infected with IBD virus . Bursae were collected 4, 8, 12, 24, 36 and 48 hours and 3, 4 and 5 days post-infection . An equal number of chickens acted as appropriate controls . The colour difference between a positive and a negative reaction was clearly distinguished with the naked eye . The cut-off level between ELISA negative and ELISA positive absorbance values was estimated at mean absorbance of negative controls plus three times the standard deviation .
INTRODUCTION The double-antibody sandwich ELISA has been developed and used for detecting some infectious agents particularly shedding of avian lymphoid leucosis virus by breeding stocks of poultry (Boer et at, 1983 ; Crittenden et at, 1984) . This paper reports the development of this technique for detection of infectious bursal disease (IBD) virus in bursal suspension from artificially infected birds .
MATERIALS AND METHODS Experimental design One hundred IBD free 5-week-old white leghorn chickens were divided into two equal groups . The individual birds of one group received IBD viral (serotype I) inoculum (103 TCID50/ml) at the rate of 0 .1 ml intraocularly and intranasally. Similarly, each bird of the second group received 0 .1 ml uninfected chicken embryo fibroblast cell culture by the same route . Five birds from each of these groups were destroyed at 4, 8, 12, 24, 36 and 48 hours and 3, 4, and 5 days post-infection (PI) . Reagents Rabbit antichicken immunoglobulin (IgG) was prepared as per the method of Smith & Witter (1983) for detection of antibodies against reticuloendotheliosis virus by the *Orissa Biological Products Institute, Bhubaneswar-3 . tAuthor for correspondence .
252
BRITISH VETERINARY JOURNAL, 147, 3
ELISA technique . Horseradish peroxidase conjugate was prepared according to Levy & Sober (1960) and Miller et al. (1974) . For separation of the gammaglobulin fraction, rabbit serum raised against chicken IgG was passed through a pre-swollen diethylamine ethylcellulose (DEAE) 52 (Watman Ltd, England) column . The conjugation procedure consisted of two phases, activation of horseradish peroxidase, type VI (Sigma) to the aldehyde form by the use of 1% 2,4-fluoronitrobenzene (Sigma) and sodium periodate . The conjugation phase consisted of addition of gammaglobulin fraction of rabbit antichicken globulin to peroxidase aldehyde . The working strength of ELISA conjugate was determined (Shekarchi & Sever, 1986) . The optimum conjugate dilution that yielded the highest positive value was found to be 1 :100 and this dilution was used during the test procedure . Rabbit hyperimmune serum against IBD virus was raised by the method of Ide (1974) for fluorescent antibody test of avian encephalomyelitis . For preparation of hyperimmune serum against IBD virus in chickens, the method of Allan & McNulty (1985) was followed . ELISA-IBD antigen was prepared as per the technique of Marquardt et al (1980) . For preparation of the bursal sample for double-antibody sandwich technique, the method of Perin et al. (1986) for rapid rabies enzyme immunodiagnosis was followed . Small pieces of bursa from infected birds were homogenized in 4 volumes of phosphate buffer saline (PBS), pH 7 .4 and centrifuged at 1400 g for 30 min to eliminate gross particles . The clear supernatant was used for detection of antigen ; a similar procedure was followed for preparation of IBD negative antigen from uninfected bursa . Determination of optimal antibody and antigen dilution
A preliminary experiment was conducted using checkerboard titration to determine the optimal antibody dilution (rabbit and chicken hyperimmune serum against IBD virus) and ELISA IBD antigen . For this test, flat bottomed 96 well microtitre plates (Nunc immunoplate-IA/S Nunc, Denmark) were used . The first two vertical rows acted as substrate (S) and conjugatesubstrate (C-S) controls, respectively . Rabbit hyperimmune serum against IBD virus was diluted arbitrarily 1 :500 ; 1 :1000 ; 1 :2000 ; 1 :4000 ; 1 :8000 in carbonate-bicarbonate buffer, pH 9 .6 . For each antibody dilution, 100 µl (automatic microtitre pipette, Dynatech) were added to two vertical rows and incubated overnight at 4° C and washed six times (Shekarchi & Sever, 1986) . ELISA antigen dilutions (1 :5 ; 1 :10 ; 1 :20 ; 1 :40 in PBS-Tween) at the rate of 100 µl were added to sensitized wells, so that all antibody dilutions were tested . After incubation for 90 min at 37°C and washing, 100 µ1 of hyperimmune serum against IBD virus raised in chickens (diluted arbitrarily 1 :1000 in PBSTween with addition of 0 .5% bovine serum albumin) were dispensed in all wells except S and C-S control wells . After incubation for 150 min at 37 °C and washing, 100µl of 1 :100 dilution of conjugate with PBS-Tween plus 0 .5% bovine serum albumin were dispensed in each well except S and C-S control wells . Following incubation for 90 min at 37°C and washing, 100,ul of freshly prepared orthophenylenediamine (OPD) with hydrogen peroxide were dispensed in all wells . After incubation for 30 min at 37 ° C the colour reaction was stopped by addition of 50µl of 2 .5 M H 2 SO4 . The reaction was read both visually and with the help of an ELISA reader (Biored Model 2550 EIA) at 490 nm . The positive reaction was orange to reddish-brown in colour. There was no colour development in S and C-S controls . The dilution of rabbit hyperimmune serum and antigen giving a reading close to an optical density (OD) of 1 .0 indicated the range to be
ELISA FOR DETECTING IBD VIRUS
253
tested with the ELISA system used . In this checkerboard titration, the optimal hyperimmune rabbit anti-IBDV serum dilution and ELISA antigen dilution that yielded highest positive values were found to be 1 :8000 and 1 :10, respectively . These dilutions were used during the test procedure . A similar checkerboard titration was performed with hyperimmune chicken anti-IBD virus serum to determine the optimal dilution yielding the highest positive value which was found to be 1 :4000 . Test procedure The protocol of WHO ELISA for Detection of Rota Virus Antigen in Faeces, WHO Collaborating Centre for Reference and Research on Rota Virus, Regional Virus Laboratory, East Birmingham Hospital, UK was followed with slight modification . One hundred,ul of 1 :8000 dilution of hyperimmune serum against IBD virus raised in rabbits diluted in carbonate-bicarbonate buffer, pH 9 .6, was added to all the wells of the microtitre plate except the first vertical row of wells from A to H which acted as S control and the last vertical row of wells of EFGH which acted as C-S control . After the plates were incubated overnight at 4 °C, a plan was prepared for samples to be tested . Four vertical wells were used per sample under investigation . EFGH wells of the 10th and 11th vertical rows acted as known negative and known positive controls in which the former received 100 pl of 1 :10 dilution of IBD negative bursal antigen and the latter the same quantity of 1 :10 dilution of known bursal antigen . The sample to be tested was diluted 1 :10 in PBS-Tween and 100 pl dispensed in all the wells except the first row of vertical wells and EFGH wells of 10, 11 and 12 vertical rows of wells . After incubation overnight at 4 °C and washing, 100,ul of 1 :4000 hyperimmune serum against IBD virus raised in chickens for sandwiching the antigen was dispensed to all the wells except the S and C-S control wells . Following incubation for 90 min at 37 °C and washing, 100#1 of rabbit anti-chicken IgG-conjugate diluted 1 :100 with PBS-Tween plus 0.5% bovine serum albumin were dispensed in all wells except the S-controls . After incubation for 90 min at 37 °C and washing, 100 pl of freshly prepared OPD with hydrogen peroxide were dispensed in all wells. After incubation for 30 min at 37°C, the colour reaction was stopped by addition of 50 pl of 2 .5 M H2 SO4 . The reaction was read both visually and with the help of the ELISA reader . There was no colour development in S and C-S and negative controls . The same test procedure was repeated with 10 known negative bursal samples (1 :10 dilution) and the reaction was read with the help of the ELISA reader at 490 rim . The cut-off level between ELISA negative and ELISA positive absorbance value was estimated at mean absorbance of negative controls plus three times the standard deviation (Boer et al, 1983) .
RESULTS The control uninfected bursal suspension did not react with immunosorbent and mean absorbance of 10 such samples was found to be 0 .115 . The cut-off level between ELISA negative and positive absorbance values was found to be 0 .140 . The results of this study indicated that the bursal samples obtained from 4 to 24 hours PI revealed OD values ranging from 0.11 to 0 .12 which were well below the cut-off level, indicating the absence of viral antigen . However, from 36 to 120 hours PI, the OD values exhibited a gradual rise in trend ranging from 0 .21 to 0 .45 (Fig . 1). It was possible to correlate the OD obtained
254
BRITISH VETERINARY JOURNAL, 147, 3 0.5-
0
5h
E 0 .4 0.3 0 v c a ° 0.2 s-•- • - • 0n a a 0 .1
Positive Negative
I I I I I I I l 1 0 4 8
18
24
36
48
72
96
120
Hours PI Fig . 1 .
Double-antibody sandwich technique for detection of IBD antigen in BF .
with this technique with viral antigen content . The colour difference between negative and positive samples was clearly distinguishable with the naked eye ; a majority of the positive samples had OD values greater than 0 .21 (a distinct yellow coloration) and most of the negative samples had OD values less than 0 .11 (absence of visible coloration) .
DISCUSSION This study shows that this technique is specific, sensitive and the results are reproducible with each sample and easy to perform . Acurate results can also be achieved rapidly. Evaluation of some other diagnostic tests such as immunoperoxidase, radial immunodiffusion and fluorescent antibody techniques (Kumar, 1990) indicated that in the first two techniques, the IBD antigen was detected from 36 hours to 4 days PI whereas in the third test, it was detected from 24 hours to 4 days PI thus confirming that the doubleantibody sandwich ELISA is equally sensitive in detecting IBD antigen in bursal tissue . An ELISA reader is not absolutely necessary for interpretation of results because the colour difference between negative and positive specimens can be clearly distinguished and the test can be performed under field conditions in India . This technique can be readily employed where there are no facilities of costly fluorescent microscopes and expertise . This test has an added advantage over the indirect ELISA because purification of the virus is not necessary . Therefore, we consider this test to be a useful tool under Indian conditions for routine laboratory diagnosis of IBD and epidemiological studies when numerous specimens have to be examined, thereby eliminating the need for conventional cumbersome virus isolation and identification methods .
ACKNOWLEDGEMENTS The senior author thanks the Indian Council of Agricultural Research, New Delhi, for awarding a senior fellowship for continuing PhD programme ; Dean of Faculty, for
ELISA FOR DETECTING IBD VIRUS
255
providing facilities ; and Dr T. N . Naik, National Institute of Cholera and Enteric Diseases, Calcutta, for help rendered .
REFERENCES ALLAN, G . M . & McNULTY, M . S . (1985) . Avian Pathology 14,449. BOER, G . F . D ., GIELKENS, A. L. J ., HARTOG, L . & BOERRIGTER, H . M . (1983) . Avian Pathology 12,447 . CRITTENDEN, L . B ., SMITH, E . J . & OKAZAKI, W . C . (1984) . Poultry Science 63, 492 . IDE, P. R . (1974) . Canadian journal of Comparative Medicine 38, 49 . KUMAR, A . (1990). PhD Thesis, OUAT, Bhubaneswar . LEVY, H . B . &SOBER, H . A . (1960) . Proceedings of the Society for Experimental Biology and Medicine 103,
250 . MARQUARDT,
W . W ., JOHNSON, R . B ., ODENWALD, W . F . &
SCHLOTHOBER,
B . A . (1980) . Avian Diseases
24,375 . H ., KARNOVSKY, M . J . & DIAMANDOPOULOS, G . (1974) . Proceedings of the Society for Experimental Biology and Medicine 146, 432 . PERRIN, P ., ROLLIN, P . E . & SUREAU, P . (1986) . Journal of Biological Standardisation 14, 217 . SHEKARCHI, I . C . & SEVER, J . L . (1986). Clinical Virology Manual eds S . Spector& G . J . Lancz . New MILLER, M .
York : Elsevier Science Publishing Company, Inc . SMITH, E . J . & WITTER, R. L . (1983) . Avian Diseases 27,
225 .
(Accepted for publication 16 August 1990)
DOUBLE-ANTIBODY SANDWICH ELISA FOR DETECTION OF INFECTIOUS BURSAL DISEASE VIRUS
AMIYA KUMAR* and A . T. RAOt Department of Pathology, Orissa Veterinary College, Bhubaneswar-751003, India SUMMARY A double-antibody sandwich ELISA was employed for detection of IBD virus in bursal suspension . Fifty IBD-free white leghorn chickens aged 5 weeks were experimentally infected with IBD virus . Bursae were collected 4, 8, 12, 24, 36 and 48 hours and 3, 4 and 5 days post-infection . An equal number of chickens acted as appropriate controls . The colour difference between a positive and a negative reaction was clearly distinguished with the naked eye . The cut-off level between ELISA negative and ELISA positive absorbance values was estimated at mean absorbance of negative controls plus three times the standard deviation .
INTRODUCTION The double-antibody sandwich ELISA has been developed and used for detecting some infectious agents particularly shedding of avian lymphoid leucosis virus by breeding stocks of poultry (Boer et at, 1983 ; Crittenden et at, 1984) . This paper reports the development of this technique for detection of infectious bursal disease (IBD) virus in bursal suspension from artificially infected birds .
MATERIALS AND METHODS Experimental design One hundred IBD free 5-week-old white leghorn chickens were divided into two equal groups . The individual birds of one group received IBD viral (serotype I) inoculum (103 TCID50/ml) at the rate of 0 .1 ml intraocularly and intranasally. Similarly, each bird of the second group received 0 .1 ml uninfected chicken embryo fibroblast cell culture by the same route . Five birds from each of these groups were destroyed at 4, 8, 12, 24, 36 and 48 hours and 3, 4, and 5 days post-infection (PI) . Reagents Rabbit antichicken immunoglobulin (IgG) was prepared as per the method of Smith & Witter (1983) for detection of antibodies against reticuloendotheliosis virus by the *Orissa Biological Products Institute, Bhubaneswar-3 . tAuthor for correspondence .
252
BRITISH VETERINARY JOURNAL, 147, 3
ELISA technique . Horseradish peroxidase conjugate was prepared according to Levy & Sober (1960) and Miller et al. (1974) . For separation of the gammaglobulin fraction, rabbit serum raised against chicken IgG was passed through a pre-swollen diethylamine ethylcellulose (DEAE) 52 (Watman Ltd, England) column . The conjugation procedure consisted of two phases, activation of horseradish peroxidase, type VI (Sigma) to the aldehyde form by the use of 1% 2,4-fluoronitrobenzene (Sigma) and sodium periodate . The conjugation phase consisted of addition of gammaglobulin fraction of rabbit antichicken globulin to peroxidase aldehyde . The working strength of ELISA conjugate was determined (Shekarchi & Sever, 1986) . The optimum conjugate dilution that yielded the highest positive value was found to be 1 :100 and this dilution was used during the test procedure . Rabbit hyperimmune serum against IBD virus was raised by the method of Ide (1974) for fluorescent antibody test of avian encephalomyelitis . For preparation of hyperimmune serum against IBD virus in chickens, the method of Allan & McNulty (1985) was followed . ELISA-IBD antigen was prepared as per the technique of Marquardt et al (1980) . For preparation of the bursal sample for double-antibody sandwich technique, the method of Perin et al. (1986) for rapid rabies enzyme immunodiagnosis was followed . Small pieces of bursa from infected birds were homogenized in 4 volumes of phosphate buffer saline (PBS), pH 7 .4 and centrifuged at 1400 g for 30 min to eliminate gross particles . The clear supernatant was used for detection of antigen ; a similar procedure was followed for preparation of IBD negative antigen from uninfected bursa . Determination of optimal antibody and antigen dilution
A preliminary experiment was conducted using checkerboard titration to determine the optimal antibody dilution (rabbit and chicken hyperimmune serum against IBD virus) and ELISA IBD antigen . For this test, flat bottomed 96 well microtitre plates (Nunc immunoplate-IA/S Nunc, Denmark) were used . The first two vertical rows acted as substrate (S) and conjugatesubstrate (C-S) controls, respectively . Rabbit hyperimmune serum against IBD virus was diluted arbitrarily 1 :500 ; 1 :1000 ; 1 :2000 ; 1 :4000 ; 1 :8000 in carbonate-bicarbonate buffer, pH 9 .6 . For each antibody dilution, 100 µl (automatic microtitre pipette, Dynatech) were added to two vertical rows and incubated overnight at 4° C and washed six times (Shekarchi & Sever, 1986) . ELISA antigen dilutions (1 :5 ; 1 :10 ; 1 :20 ; 1 :40 in PBS-Tween) at the rate of 100 µl were added to sensitized wells, so that all antibody dilutions were tested . After incubation for 90 min at 37°C and washing, 100 µ1 of hyperimmune serum against IBD virus raised in chickens (diluted arbitrarily 1 :1000 in PBSTween with addition of 0 .5% bovine serum albumin) were dispensed in all wells except S and C-S control wells . After incubation for 150 min at 37 °C and washing, 100µl of 1 :100 dilution of conjugate with PBS-Tween plus 0 .5% bovine serum albumin were dispensed in each well except S and C-S control wells . Following incubation for 90 min at 37°C and washing, 100,ul of freshly prepared orthophenylenediamine (OPD) with hydrogen peroxide were dispensed in all wells . After incubation for 30 min at 37 ° C the colour reaction was stopped by addition of 50µl of 2 .5 M H 2 SO4 . The reaction was read both visually and with the help of an ELISA reader (Biored Model 2550 EIA) at 490 nm . The positive reaction was orange to reddish-brown in colour. There was no colour development in S and C-S controls . The dilution of rabbit hyperimmune serum and antigen giving a reading close to an optical density (OD) of 1 .0 indicated the range to be
ELISA FOR DETECTING IBD VIRUS
253
tested with the ELISA system used . In this checkerboard titration, the optimal hyperimmune rabbit anti-IBDV serum dilution and ELISA antigen dilution that yielded highest positive values were found to be 1 :8000 and 1 :10, respectively . These dilutions were used during the test procedure . A similar checkerboard titration was performed with hyperimmune chicken anti-IBD virus serum to determine the optimal dilution yielding the highest positive value which was found to be 1 :4000 . Test procedure The protocol of WHO ELISA for Detection of Rota Virus Antigen in Faeces, WHO Collaborating Centre for Reference and Research on Rota Virus, Regional Virus Laboratory, East Birmingham Hospital, UK was followed with slight modification . One hundred,ul of 1 :8000 dilution of hyperimmune serum against IBD virus raised in rabbits diluted in carbonate-bicarbonate buffer, pH 9 .6, was added to all the wells of the microtitre plate except the first vertical row of wells from A to H which acted as S control and the last vertical row of wells of EFGH which acted as C-S control . After the plates were incubated overnight at 4 °C, a plan was prepared for samples to be tested . Four vertical wells were used per sample under investigation . EFGH wells of the 10th and 11th vertical rows acted as known negative and known positive controls in which the former received 100 pl of 1 :10 dilution of IBD negative bursal antigen and the latter the same quantity of 1 :10 dilution of known bursal antigen . The sample to be tested was diluted 1 :10 in PBS-Tween and 100 pl dispensed in all the wells except the first row of vertical wells and EFGH wells of 10, 11 and 12 vertical rows of wells . After incubation overnight at 4 °C and washing, 100,ul of 1 :4000 hyperimmune serum against IBD virus raised in chickens for sandwiching the antigen was dispensed to all the wells except the S and C-S control wells . Following incubation for 90 min at 37 °C and washing, 100#1 of rabbit anti-chicken IgG-conjugate diluted 1 :100 with PBS-Tween plus 0.5% bovine serum albumin were dispensed in all wells except the S-controls . After incubation for 90 min at 37 °C and washing, 100 pl of freshly prepared OPD with hydrogen peroxide were dispensed in all wells. After incubation for 30 min at 37°C, the colour reaction was stopped by addition of 50 pl of 2 .5 M H2 SO4 . The reaction was read both visually and with the help of the ELISA reader . There was no colour development in S and C-S and negative controls . The same test procedure was repeated with 10 known negative bursal samples (1 :10 dilution) and the reaction was read with the help of the ELISA reader at 490 rim . The cut-off level between ELISA negative and ELISA positive absorbance value was estimated at mean absorbance of negative controls plus three times the standard deviation (Boer et al, 1983) .
RESULTS The control uninfected bursal suspension did not react with immunosorbent and mean absorbance of 10 such samples was found to be 0 .115 . The cut-off level between ELISA negative and positive absorbance values was found to be 0 .140 . The results of this study indicated that the bursal samples obtained from 4 to 24 hours PI revealed OD values ranging from 0.11 to 0 .12 which were well below the cut-off level, indicating the absence of viral antigen . However, from 36 to 120 hours PI, the OD values exhibited a gradual rise in trend ranging from 0 .21 to 0 .45 (Fig . 1). It was possible to correlate the OD obtained
254
BRITISH VETERINARY JOURNAL, 147, 3 0.5-
0
5h
E 0 .4 0.3 0 v c a ° 0.2 s-•- • - • 0n a a 0 .1
Positive Negative
I I I I I I I l 1 0 4 8
18
24
36
48
72
96
120
Hours PI Fig . 1 .
Double-antibody sandwich technique for detection of IBD antigen in BF .
with this technique with viral antigen content . The colour difference between negative and positive samples was clearly distinguishable with the naked eye ; a majority of the positive samples had OD values greater than 0 .21 (a distinct yellow coloration) and most of the negative samples had OD values less than 0 .11 (absence of visible coloration) .
DISCUSSION This study shows that this technique is specific, sensitive and the results are reproducible with each sample and easy to perform . Acurate results can also be achieved rapidly. Evaluation of some other diagnostic tests such as immunoperoxidase, radial immunodiffusion and fluorescent antibody techniques (Kumar, 1990) indicated that in the first two techniques, the IBD antigen was detected from 36 hours to 4 days PI whereas in the third test, it was detected from 24 hours to 4 days PI thus confirming that the doubleantibody sandwich ELISA is equally sensitive in detecting IBD antigen in bursal tissue . An ELISA reader is not absolutely necessary for interpretation of results because the colour difference between negative and positive specimens can be clearly distinguished and the test can be performed under field conditions in India . This technique can be readily employed where there are no facilities of costly fluorescent microscopes and expertise . This test has an added advantage over the indirect ELISA because purification of the virus is not necessary . Therefore, we consider this test to be a useful tool under Indian conditions for routine laboratory diagnosis of IBD and epidemiological studies when numerous specimens have to be examined, thereby eliminating the need for conventional cumbersome virus isolation and identification methods .
ACKNOWLEDGEMENTS The senior author thanks the Indian Council of Agricultural Research, New Delhi, for awarding a senior fellowship for continuing PhD programme ; Dean of Faculty, for
ELISA FOR DETECTING IBD VIRUS
255
providing facilities ; and Dr T. N . Naik, National Institute of Cholera and Enteric Diseases, Calcutta, for help rendered .
REFERENCES ALLAN, G . M . & McNULTY, M . S . (1985) . Avian Pathology 14,449. BOER, G . F . D ., GIELKENS, A. L. J ., HARTOG, L . & BOERRIGTER, H . M . (1983) . Avian Pathology 12,447 . CRITTENDEN, L . B ., SMITH, E . J . & OKAZAKI, W . C . (1984) . Poultry Science 63, 492 . IDE, P. R . (1974) . Canadian journal of Comparative Medicine 38, 49 . KUMAR, A . (1990). PhD Thesis, OUAT, Bhubaneswar . LEVY, H . B . &SOBER, H . A . (1960) . Proceedings of the Society for Experimental Biology and Medicine 103,
250 . MARQUARDT,
W . W ., JOHNSON, R . B ., ODENWALD, W . F . &
SCHLOTHOBER,
B . A . (1980) . Avian Diseases
24,375 . H ., KARNOVSKY, M . J . & DIAMANDOPOULOS, G . (1974) . Proceedings of the Society for Experimental Biology and Medicine 146, 432 . PERRIN, P ., ROLLIN, P . E . & SUREAU, P . (1986) . Journal of Biological Standardisation 14, 217 . SHEKARCHI, I . C . & SEVER, J . L . (1986). Clinical Virology Manual eds S . Spector& G . J . Lancz . New MILLER, M .
York : Elsevier Science Publishing Company, Inc . SMITH, E . J . & WITTER, R. L . (1983) . Avian Diseases 27,
225 .
(Accepted for publication 16 August 1990)