J. Vet. Med. B 37, 753-759 (1990) 0 1990 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 093 1- 1793
Department of Bacteriology, Central Veterinay Institute, Lelystad
Enzyme Immunoassay Using Mouse Monoclonal Anti-bovine Antibodies for the Detection of Brucella abortus Antibodies in Cow Milk 2. BERCOVICH and R. TAAIJKE Address of authors: Central Veterinary Institute, Department of Bacteriology, P. 0. Box 65, 8200 AB Lelystad, The Netherlands
With 3 tables (Received for publication April 18, 1990)
Summary Individual milk samples and artificially constructed tank milk samples from cows with naturally occurring brucellosis were examined by the enzyme-linked immunosorbent assay (ELISA) using sonicated B. ubortus S-99 antigen, and mouse monoclonal anti-bovine IgM, IgA, and IgG, conjugates. ELISA results were compared with the results of the milk ring test using either 1 ml milk (MRT-1) o r 8 ml milk (MRT-X). The ELISA using mouse monoclonal anti-bovine IgG, conjugate was sensitive and specific. In testing individual milk samples and constructed tank milk samples containing milk with low titers in the MRT-I the ELISA was superior to the MRT-1, and MRT-S. In testing other milk samples, the ELISA was as sensitive or slightly less sensitive than the MRT-B. From a total of 5,910 milk samples collected from cows free from brucellosis, only 24 (0.4 %) samples tested positive in the ELISA. All 500 tank milk samples collected from farms negative for brucellosis tested negative in the ELISA. We concluded that the ELISA is a good substitute for the MRT-I to detect antibodies against Brucellu in milk from individual cows. When tank milk is tested for antibodies against Brucellu, however. both the MRT-8 and the ELISA should be used.
Introduction Although Brucella abortus has long been eradicated in cattle in the Netherlands, infected cattle occasionally have been detected on farms that purchased imported cattle. The milk ring test, which uses 1 ml milk (MRT-I), is the principal test for surveillance of brucellosis on dairy farms in many countries. Certain kinds of milk however, may react non-specifically in the MRT-I. Milk containing colostrum, milk examined at the end of the lactation period, milk from cows with hormonal disorders, and milk which fat globules d o not cluster (KEER et al., 1959; MCCAUGNY,1972; BERCOVICHand MOERMAN,1979; CORBEL et al., 1984; PATTERSON et al., 1976). Moreover, the MRT-1 is inadequate to detect antibodies against Brucellu in tank milk (HILLand CREMERS, 1967). Although the sensitivity of the MRT-1 was considerably improved after the introduction of the MRT and LAGENDIJK, 1978), 3 of the reactions are still nonthat uses 8 ml milk (BERCOVICH specific. U.S. C y y r i g h r C l c x ~ n c eCentcr Code
Statement:
0931 - 1793/90/3710-0753$02.50/0
754
BERCOVICH and TAAIJKE
T h e enzyme-linked immunosorbent assay (ELISA) used to detect antibodies against Brucella in milk f r o m individual cows is sensitive and specific, and m a y also be suitable for the detection of antibodies against Brucella in tank milk (THOENet al., 1979; HECK et al., 1980; BORAKER,1981; OLIVER and COOPER, 1981; FORSCHNER et al., 1989). FORSCHNER and BUNCER(1986) improved the sensitivity of the ELISA by concentrating milk serum gammaglobulins with ammonium sulfate and were able t o detect antibodies against Brucella in tank milk comprising milk samples f r o m 50 cows or more. Routine use of this procedure o n a large scale is impractical. In the assay reported here w e used mouse monoclonal anti-bovine IgM, IgA, and IgG, conjugates t o detect Brucella antibodies in milk collected f r o m individual cows and in artificially constructed tank milk.
Material and Methods
Milk and serum samples Milk was collected from two cows with naturally occurring brucellosis. Both cows tested positive to serologic tests, the delayed-type hypersensitivity test performed according to BERCOVICH et al. (1989), and bacteriologic examination performed according to BERCOVICH and TER LAAK(1990). Milk from cow 4473 tested negative and milk from cow 4474 tested positive in the MRT-1. To determine the sensitivity of the ELISA, we prepared whey samples from 39 milk samples that were positive or negative in the MRT-1 and the ELISA. The whey samples were examined by the serum agglutination test, complement fixation test, and Coombs test. Tank milk samples were constructed as follows. Milk samples from 14 cows positive for Brucella in at least one serologic test were examined with the MRT-1 and the ELISA to establish the titers. The milk samples were then diluted to mimic tank milk samples. Milk from cows 4473 and 4474 was diluted to construct samples “representing” cow’s milk with MRT-1 titers of 2, 4, 8, or 16. Tank milk samples were constructed by further diluting these samples. All dilutions were made with milk that tested negative in the MRT-1. The constructed tank milk samples were examined by the MRT-8 and the ELISA. To determine the specificity of the ELISA, we examined the following milk samples in an ELISA using mouse monoclonal anti-bovine IgG,: 4,072 milk samples from healthy cows, 943 quarter milk samples from cows with mastitis, 35 milk samples from cows treated with prostaglandins for hormonal disorders, and 500 tank milk samples. An additional 860 milk samples were examined with the MRT-1 and the ELISA. All samples were collected from farms that were officially recognized as free of brucellosis. Milk ring test Milk samples from individual cows were examined with the MRT-1 according to HILL(1966). Constructed tank milk samples were examined with the MRT that uses 8 ml milk MRT-8 according to BERCOVICH and LACENDIJK (1978). The antigen was produced and standardized at the Central Veterinary Institute, Lelystad. Serologic examination of whey and serum Whey was prepared from milk as follows: 5 ml of each milk sample was centrifuged for 20 min at 1,300 g. After the fat was removed, 2 drops of rennet and 2 drops of saturated CaClz were added to each sample. The sample was then mixed and incubated in a 37°C waterbath for 30 min. The whey was collected by centrifugation. Whey and serum samples were examined serologically as described earlier by BERCOVICH et al. (1989).
-
ELISA Antigen Brucella abortus strain 99 was used to produce the antigen. The bacteria were grown on tryptose agar in Roux flasks for 4 days at 37°C. The bacteria were harvested with sterile saline, autoclaved for 25 min at 121 ’C, cooled to room temperature, and filtered through a sterile gauze to remove agar particles. The bacteria were then disrupted by sonification for 20 min at 20Kc/s on a 500W disintegrator. The resulting suspension was stored in stock solution at 4-6 “C. To prevent bacterial contamination, 0.1 % formaldehyde (final concentration) was added.
Enzyme Immunoassay Using Mouse Monoclonal Anti-bovine Antibodies
755
Preparation of antigen-coated plates The wells of the ELISA plates were filled with 0.1 ml antigen diluted in 0.01 M phosphatebuffered saline (PBS) pH6.3, sealed, and incubated overnight at 4°C. The coated plates were stored at - 20 "C until used. The optimal antigen concentration for coating the ELISA plates was determined by a block titration against the second international anti-Brucella standard serum. Antigen and conjugate concentrations were adjusted to detect 0.0025 -0.005 Brucella antibody unit per well. Conjugates and substrate Mouse monoclonal anti-bovine IgM (CVI 17.4.lab), IgA (CVI 15.8.la), and IgGl (CVI 15.8.1) antibodies labelled with horseradish peroxidase were used as conjugates. The monoclonal antibodies (1984). The conjugated monoclonal antibodies were produced by Dr. D. VAN ZAANEand J. IJZERMAN from the Bacteriology Department of the Central were kindly supplied by Dr. F. M. VAN ZIJDERVELD Veterinary Institute, Lelystad. The conjugates were diluted in 0.5M PBS (pH 7.2), consisting of 0.12 g KH,PO,; 16 g Na2HP04.2H,O; 29.2 g NaCI; and 0.5 ml Tween 20 dissolved in 1 liter distilled water. The working dilutions were determined by block titration. The substrate prepared by dissolving 1 g 5-amino-2-hydroxybenzoicacid (Merck, art. 819019) in 1 liter 0.01 M PBS containing 0.04 g Na2EDTA had a p H of 5.9-6.0. The substrate solution was divided into 9ml amounts and kept at -20 "C until used. Just before use, 1 ml of 0.1 YO H,02 solution was added to 9 ml defrosted substrate solution. ELISA procedure The antigen in the coated ELISA plates was defrosted in a 37°C waterbath and discarded. The plates were washed five times with a total amount of 500ml washing solution, consisting of demineralized water and 0.05 % (v/v) Tween 20. Appropriate controls were examined on each plate in columns 1 to 3. Milk samples were examined in columns 4 to 12. Two-hundred microliters of each sample was dispensed in a well in row A. The other wells were filled with 0.1 mlO.01 M PBS (pH 7.2) containing 0.05 % (v/v) Tween 20, and twofold dilutions of control and test samples were made. Samples used to evaluate the specificity of the ELISA were undiluted and were examined in duplicate. After 1 h incubation at 3 7 T , the plates were washed as described above, and 0.1 ml of appropriate mouse monoclonal anti-bovine conjugate, diluted in 0.5 M PBS (pH 7.2), was added to each well. After 1 h incubation at 37"C, the plates were washed again as described above, and 0.1 ml substrate solution containing 0.1 H202was added to each well. After overnight incubation at room temperature, the plates were shaken on a micro-shaker, and the results were recorded on an Easy reader EAR 400 at 450 nm (SLT-Labinstruments, Austria). When testing milk from individual con \. we chose as a cut off point an extinction value equal to the extinction value of 0.005 Brucella antibody unit per well. To increase the sensitivity of the ELISA in detecting antibodies against Brucella in tank milk, we chose as a cut off point an extinction value equal to the extinction value of 0.0025 Brucella antibody unit per well.
Results W h e n milk samples f r o m cows 4473 and 4474 were tested, mouse monoclonal antibovine IgM and IgA conjugates yield titers lower than the anti-bovine IgGl conjugate, T h e I g G l titer in milk f r o m c o w 4473 was 1,200 whereas the I g G l titer in milk f r o m cow 4474 was 300. A mixture of the three conjugates did not yield titers higher than I g G l conjugate alone. Sensitivity of the ELISA. W h e y samples made from milk that tested negative in t h e ELISA tested also negative in serologic tests. W h e y samples made from milk with low titers in t h e ELISA tested positive in at least o n e of the serologic tests. Whey samples made f r o m milk that tested positive in the ELISA were also positive in serologic tests (Table 1). W h e n constructed tank milk samples f r o m cows w i t h naturally occurring brucellosis were tested, the ELISA detected antibodies against Brucella w h e n b o t h the MRT-1 a n d MRT-8 w e r e negative (Table 2). W h e n constructed tank milk samples containing cow's milk with a high concentration of I g G l were tested, ELISA titers were higher than MRT-8 titers (Table 3, c o w 4473). W h e n constructed tank milk samples containing cow's milk with a low concentration of I g G l were tested, ELISA titers were slightly lower than MRT-8 titers (Table 3, c o w 4474).
756
BERCOVICH and TAAIJKE
Table 1. Comparison of serologic test titers and IgG,-ELISA titers of whey and milk collected from cows with naturally occurring brucellosis
Cow No.
SAT;>
Whey titers CFT
Milk titers Coombs
ELISA
neg. 7.5 7.5 7.5 15 30 60 ND ND ND ND ND ND N I) ND ND ND 240 960
neg. 2 4 8 16 64 32 128 128 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 32 32
~
1-2 3-6 7 8-9 10 11-13 14 15 16 17 18 19-22 23-29 30 31 -32 33-36 37 38 39 .'
neg. neg. neg. 7.5 7.5 7.5 15 neg. 15 15 30 30 30 60 60 60 60 120 120
< < < <
Department of Bacteriology, Central Veterinay Institute, Lelystad
Enzyme Immunoassay Using Mouse Monoclonal Anti-bovine Antibodies for the Detection of Brucella abortus Antibodies in Cow Milk 2. BERCOVICH and R. TAAIJKE Address of authors: Central Veterinary Institute, Department of Bacteriology, P. 0. Box 65, 8200 AB Lelystad, The Netherlands
With 3 tables (Received for publication April 18, 1990)
Summary Individual milk samples and artificially constructed tank milk samples from cows with naturally occurring brucellosis were examined by the enzyme-linked immunosorbent assay (ELISA) using sonicated B. ubortus S-99 antigen, and mouse monoclonal anti-bovine IgM, IgA, and IgG, conjugates. ELISA results were compared with the results of the milk ring test using either 1 ml milk (MRT-1) o r 8 ml milk (MRT-X). The ELISA using mouse monoclonal anti-bovine IgG, conjugate was sensitive and specific. In testing individual milk samples and constructed tank milk samples containing milk with low titers in the MRT-I the ELISA was superior to the MRT-1, and MRT-S. In testing other milk samples, the ELISA was as sensitive or slightly less sensitive than the MRT-B. From a total of 5,910 milk samples collected from cows free from brucellosis, only 24 (0.4 %) samples tested positive in the ELISA. All 500 tank milk samples collected from farms negative for brucellosis tested negative in the ELISA. We concluded that the ELISA is a good substitute for the MRT-I to detect antibodies against Brucellu in milk from individual cows. When tank milk is tested for antibodies against Brucellu, however. both the MRT-8 and the ELISA should be used.
Introduction Although Brucella abortus has long been eradicated in cattle in the Netherlands, infected cattle occasionally have been detected on farms that purchased imported cattle. The milk ring test, which uses 1 ml milk (MRT-I), is the principal test for surveillance of brucellosis on dairy farms in many countries. Certain kinds of milk however, may react non-specifically in the MRT-I. Milk containing colostrum, milk examined at the end of the lactation period, milk from cows with hormonal disorders, and milk which fat globules d o not cluster (KEER et al., 1959; MCCAUGNY,1972; BERCOVICHand MOERMAN,1979; CORBEL et al., 1984; PATTERSON et al., 1976). Moreover, the MRT-1 is inadequate to detect antibodies against Brucellu in tank milk (HILLand CREMERS, 1967). Although the sensitivity of the MRT-1 was considerably improved after the introduction of the MRT and LAGENDIJK, 1978), 3 of the reactions are still nonthat uses 8 ml milk (BERCOVICH specific. U.S. C y y r i g h r C l c x ~ n c eCentcr Code
Statement:
0931 - 1793/90/3710-0753$02.50/0
754
BERCOVICH and TAAIJKE
T h e enzyme-linked immunosorbent assay (ELISA) used to detect antibodies against Brucella in milk f r o m individual cows is sensitive and specific, and m a y also be suitable for the detection of antibodies against Brucella in tank milk (THOENet al., 1979; HECK et al., 1980; BORAKER,1981; OLIVER and COOPER, 1981; FORSCHNER et al., 1989). FORSCHNER and BUNCER(1986) improved the sensitivity of the ELISA by concentrating milk serum gammaglobulins with ammonium sulfate and were able t o detect antibodies against Brucella in tank milk comprising milk samples f r o m 50 cows or more. Routine use of this procedure o n a large scale is impractical. In the assay reported here w e used mouse monoclonal anti-bovine IgM, IgA, and IgG, conjugates t o detect Brucella antibodies in milk collected f r o m individual cows and in artificially constructed tank milk.
Material and Methods
Milk and serum samples Milk was collected from two cows with naturally occurring brucellosis. Both cows tested positive to serologic tests, the delayed-type hypersensitivity test performed according to BERCOVICH et al. (1989), and bacteriologic examination performed according to BERCOVICH and TER LAAK(1990). Milk from cow 4473 tested negative and milk from cow 4474 tested positive in the MRT-1. To determine the sensitivity of the ELISA, we prepared whey samples from 39 milk samples that were positive or negative in the MRT-1 and the ELISA. The whey samples were examined by the serum agglutination test, complement fixation test, and Coombs test. Tank milk samples were constructed as follows. Milk samples from 14 cows positive for Brucella in at least one serologic test were examined with the MRT-1 and the ELISA to establish the titers. The milk samples were then diluted to mimic tank milk samples. Milk from cows 4473 and 4474 was diluted to construct samples “representing” cow’s milk with MRT-1 titers of 2, 4, 8, or 16. Tank milk samples were constructed by further diluting these samples. All dilutions were made with milk that tested negative in the MRT-1. The constructed tank milk samples were examined by the MRT-8 and the ELISA. To determine the specificity of the ELISA, we examined the following milk samples in an ELISA using mouse monoclonal anti-bovine IgG,: 4,072 milk samples from healthy cows, 943 quarter milk samples from cows with mastitis, 35 milk samples from cows treated with prostaglandins for hormonal disorders, and 500 tank milk samples. An additional 860 milk samples were examined with the MRT-1 and the ELISA. All samples were collected from farms that were officially recognized as free of brucellosis. Milk ring test Milk samples from individual cows were examined with the MRT-1 according to HILL(1966). Constructed tank milk samples were examined with the MRT that uses 8 ml milk MRT-8 according to BERCOVICH and LACENDIJK (1978). The antigen was produced and standardized at the Central Veterinary Institute, Lelystad. Serologic examination of whey and serum Whey was prepared from milk as follows: 5 ml of each milk sample was centrifuged for 20 min at 1,300 g. After the fat was removed, 2 drops of rennet and 2 drops of saturated CaClz were added to each sample. The sample was then mixed and incubated in a 37°C waterbath for 30 min. The whey was collected by centrifugation. Whey and serum samples were examined serologically as described earlier by BERCOVICH et al. (1989).
-
ELISA Antigen Brucella abortus strain 99 was used to produce the antigen. The bacteria were grown on tryptose agar in Roux flasks for 4 days at 37°C. The bacteria were harvested with sterile saline, autoclaved for 25 min at 121 ’C, cooled to room temperature, and filtered through a sterile gauze to remove agar particles. The bacteria were then disrupted by sonification for 20 min at 20Kc/s on a 500W disintegrator. The resulting suspension was stored in stock solution at 4-6 “C. To prevent bacterial contamination, 0.1 % formaldehyde (final concentration) was added.
Enzyme Immunoassay Using Mouse Monoclonal Anti-bovine Antibodies
755
Preparation of antigen-coated plates The wells of the ELISA plates were filled with 0.1 ml antigen diluted in 0.01 M phosphatebuffered saline (PBS) pH6.3, sealed, and incubated overnight at 4°C. The coated plates were stored at - 20 "C until used. The optimal antigen concentration for coating the ELISA plates was determined by a block titration against the second international anti-Brucella standard serum. Antigen and conjugate concentrations were adjusted to detect 0.0025 -0.005 Brucella antibody unit per well. Conjugates and substrate Mouse monoclonal anti-bovine IgM (CVI 17.4.lab), IgA (CVI 15.8.la), and IgGl (CVI 15.8.1) antibodies labelled with horseradish peroxidase were used as conjugates. The monoclonal antibodies (1984). The conjugated monoclonal antibodies were produced by Dr. D. VAN ZAANEand J. IJZERMAN from the Bacteriology Department of the Central were kindly supplied by Dr. F. M. VAN ZIJDERVELD Veterinary Institute, Lelystad. The conjugates were diluted in 0.5M PBS (pH 7.2), consisting of 0.12 g KH,PO,; 16 g Na2HP04.2H,O; 29.2 g NaCI; and 0.5 ml Tween 20 dissolved in 1 liter distilled water. The working dilutions were determined by block titration. The substrate prepared by dissolving 1 g 5-amino-2-hydroxybenzoicacid (Merck, art. 819019) in 1 liter 0.01 M PBS containing 0.04 g Na2EDTA had a p H of 5.9-6.0. The substrate solution was divided into 9ml amounts and kept at -20 "C until used. Just before use, 1 ml of 0.1 YO H,02 solution was added to 9 ml defrosted substrate solution. ELISA procedure The antigen in the coated ELISA plates was defrosted in a 37°C waterbath and discarded. The plates were washed five times with a total amount of 500ml washing solution, consisting of demineralized water and 0.05 % (v/v) Tween 20. Appropriate controls were examined on each plate in columns 1 to 3. Milk samples were examined in columns 4 to 12. Two-hundred microliters of each sample was dispensed in a well in row A. The other wells were filled with 0.1 mlO.01 M PBS (pH 7.2) containing 0.05 % (v/v) Tween 20, and twofold dilutions of control and test samples were made. Samples used to evaluate the specificity of the ELISA were undiluted and were examined in duplicate. After 1 h incubation at 3 7 T , the plates were washed as described above, and 0.1 ml of appropriate mouse monoclonal anti-bovine conjugate, diluted in 0.5 M PBS (pH 7.2), was added to each well. After 1 h incubation at 37"C, the plates were washed again as described above, and 0.1 ml substrate solution containing 0.1 H202was added to each well. After overnight incubation at room temperature, the plates were shaken on a micro-shaker, and the results were recorded on an Easy reader EAR 400 at 450 nm (SLT-Labinstruments, Austria). When testing milk from individual con \. we chose as a cut off point an extinction value equal to the extinction value of 0.005 Brucella antibody unit per well. To increase the sensitivity of the ELISA in detecting antibodies against Brucella in tank milk, we chose as a cut off point an extinction value equal to the extinction value of 0.0025 Brucella antibody unit per well.
Results W h e n milk samples f r o m cows 4473 and 4474 were tested, mouse monoclonal antibovine IgM and IgA conjugates yield titers lower than the anti-bovine IgGl conjugate, T h e I g G l titer in milk f r o m c o w 4473 was 1,200 whereas the I g G l titer in milk f r o m cow 4474 was 300. A mixture of the three conjugates did not yield titers higher than I g G l conjugate alone. Sensitivity of the ELISA. W h e y samples made from milk that tested negative in t h e ELISA tested also negative in serologic tests. W h e y samples made from milk with low titers in t h e ELISA tested positive in at least o n e of the serologic tests. Whey samples made f r o m milk that tested positive in the ELISA were also positive in serologic tests (Table 1). W h e n constructed tank milk samples f r o m cows w i t h naturally occurring brucellosis were tested, the ELISA detected antibodies against Brucella w h e n b o t h the MRT-1 a n d MRT-8 w e r e negative (Table 2). W h e n constructed tank milk samples containing cow's milk with a high concentration of I g G l were tested, ELISA titers were higher than MRT-8 titers (Table 3, c o w 4473). W h e n constructed tank milk samples containing cow's milk with a low concentration of I g G l were tested, ELISA titers were slightly lower than MRT-8 titers (Table 3, c o w 4474).
756
BERCOVICH and TAAIJKE
Table 1. Comparison of serologic test titers and IgG,-ELISA titers of whey and milk collected from cows with naturally occurring brucellosis
Cow No.
SAT;>
Whey titers CFT
Milk titers Coombs
ELISA
neg. 7.5 7.5 7.5 15 30 60 ND ND ND ND ND ND N I) ND ND ND 240 960
neg. 2 4 8 16 64 32 128 128 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 32 32
~
1-2 3-6 7 8-9 10 11-13 14 15 16 17 18 19-22 23-29 30 31 -32 33-36 37 38 39 .'
neg. neg. neg. 7.5 7.5 7.5 15 neg. 15 15 30 30 30 60 60 60 60 120 120
< < < <
