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Titration of allergenicity using a passive cutaneous anaphylaxis inhibition test
Journal o f l m r n u n o l o g i c a l M e t h o d s , 64 (1983) 249-254
249
Elsevier JIM02816
Titration of Allergenicity using a Passive Cutaneous Anaphylaxis Inhibition Test L. B l a c k a n d M . J . F r a n c i s The Wellcome Foot and Mouth Disease Vaccine Laboratory, Pirbright, Woking, Surrey, U.K.
(Received 1 February 1983, accepted 25 May 1983)
A method for titrating allergenicity is described and validated by demonstrating that both baby hamster kidney cell lysate and hamster serum are capable of inhibiting the passive cutaneous anaphylaxis reactions produced by anti-BHK bovine serum in the skin of goats. Key words: cattle - reagins - passive cutaneous anaphylaxis - allergens
Introduction
Radioallergosorbent techniques (Wide et al., 1967) are commonly used to quantify substances which are allergenic for man. These methods, however, call for the use of specific anti-IgE sera which are seldom available for non-human species. In the past we used the passive cutaneous anaphylaxis (PCA) method to identify substances which were potentially allergenic for cattle (Black et al., 1975). This test, carried out by injecting cattle antiserum intradermally into goats, and injecting Evans blue and the suspect allergens intravenously 3 days later, was, however, not ideal for the quantitative assessment of allergenic activity. There have been reports that Prausnitz-Ktistner reactions in man could be inhibited by admixture of the antiserum with its specific allergen (Levine and Coca, 1926; Patterson and Correa, 1959) and this suggested that a method involving inhibition of PCA reactions might be useful for titrating the activity of allergens when the use of radioallergosorbent methods is precluded. In this study we aimed to validate the passive cutaneous anaphylaxis inhibition (PCA-I) method by measuring the inhibiting activity of baby hamster kidney (BHK) cell lysate on the action of its specific bovine-derived antiserum. We sought to demonstrate that inhibition endpoints are dependent on a quantitative interrelationship between the concentrations of the antiserum and the inhibitor. BHK cell lysate was selected as the antigen because previous results have demonstrated its ability to stimulate reaginic antibodies in cattle (Black, 1977a, b). We also took the opportunity of measuring the inhibition produced by dilutions 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
250 of hamster serum (HS) because allergenic cross-reactions between horse serum and epithelial cell derivatives have been previously demonstrated in the case of horse dander allergy in man (Stanworth, 1959).
Materials and Methods Animals Two white female Saanen goats about 10 months old were used. B H K cell lysate (Stoker and McPherson, 1964) Four litres of cell harvest containing 4.1 x 10 6 BHK-21 suspension culture cells/ml were centrifuged at 500 × g for 10 min and the sedimented cells were washed three times in phosphate buffered saline. The cells were then suspended in 100 ml distilled water and frozen and thawed three times by immersing in liquid nitrogen. The gross cell debris was sedimented at 1000 × g for 10 min and the supernatant clarified by spinning at 75,000 × g for 60 min. The concentration of protein in the supernatant, assessed by Bio-Rad Reagent (Bio-Rad Laboratories), was 23.6 m g / m l . The P C A - I test The goats were sedated by injecting 0.4 ml of 2% xylazine (Rompun, Bayer) intramuscularly and the hair over the chest walls and flanks was clipped. The skin on each side was marked out in a grid of 6 × 10 blocks, each about 3 cm × 4 cm. Bovine serum (No. 2358), which was known to contain anti-BHK reaginic antibodies, was made up in a doubling dilution series from 1 / 4 to 1/128 in normal saline and mixed with equal volumes of either B H K cell lysate (doubling dilutions from 1 / 8 to 1/2048) or HS (doubling dilutions from 1/1 to 1/256). (All subsequent dilutions are expressed as final dilutions after mixing.) The mixtures were left at 4°C for 24 h and then injected into the skin of the goats, 0.1 ml per block, using 1 ml plastic syringes and 26-gauge needles. The injections were administered in chequer board fashion, with the dilutions of antiserum running vertically and the dilutions of lysate or HS running horizontally (see Fig. 2). The antiserum-BHK cell lysate mixtures were injected on the left side of each animal and the antiserum-HS mixtures on the right. On each side a control series was also included comprising an antiserum dilution series mixed with an equal volume of saline only. Identical tests were performed on each of the goats to examine the reproducibility of the results. Three days later, 10 ml of 2% Evans blue dye followed by 6 ml of the B H K cell lysate were injected intravenously and, after 45 rain, the blue stains which appeared at the positive reaction sites were noted and photographed. Results
Fig. 1 is a photograph of the reactions on the right side of goat NT4 and Fig. 2 a - d represents diagrammatically all the reactions which were observed.
251
Fig. 1. The right side of goat NT4 showing the inhibitory activity of hamster serum on anti-BHK reaginic serum. For detailed annotation see Fig. 2d.
a). NT3,LHS: BHK
b~ NT4,LHS: lysate
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81
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lysate
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c~ NT3,RHS:
d~ NT4,RHS:
HS
HS
Q. C ."*" ~'..
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81 1~32 I 64
l*l*l*l*l*l.l*l,l II I°1°1°1°1°1°1-1 II II II°l°lolel°l I I I I i I°l°l°l III]
2561
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I I I I I 1 I I i
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dilutions of i n h i b i t o r s - - ~ Fig. 2. The left and right sides of goats NT3 and NT4. The reaginic serum (dilutions from top to bottom) were mixed with equal volumes of either B H K cell lysate or hamster serum (HS) (dilutions from left to right). The black dots in the blocks represent reactions. The column at the extreme right of each box contains dilutions of the reaginic antiserum mixed with saline only. (Dilutions expressed as final dilution reciprocals.)
252
I
b) Goat No.
a) Goat No. NT 3
A O v-3.0
/SBHK
m/m
ly.~.
3.0
v
1.0
I
w/8
/
BHK lys.
,.o
2.0
.1¢
NT4
/,
0 0.9
1.0
1.2
1.5
1.8
0
0.9
1.2
1.5
dilution of reaginic antiserum(log10)
1.8
--
Fig. 3. The inhibiting titres of BHK cell lysate (BHK lys.) and hamster serum (H. Ser.) on dilutions of anti-BHK reaginic serum.
Both BHK cell lysate and HS inhibited the PCA reactions and the inhibition endpoints were dependent on the dilution of the reaginic antiserum employed. For instance Fig. 2d shows that the inhibiting titre of HS on goat NT4 was 1/4, 1/8, 1/32 and 1/128 when mixed with the reaginic antiserum diluted 1/8, 1/16, 1/32 and 1/64 respectively. When reaginic antiserum dilutions were mixed with saline only, the reactions occurred to a limiting dilution of 1/32 on goat NT3 and 1/64 on goat NT4 (see Fig. 2, column at the extreme right of each grid), reflecting the different sensitivities of the animals involved. In Fig. 3a and b the inhibition produced by the BHK cell lysate and HS have been plotted against the antiserum dilution and it is evident that the BHK cell lysate was approximately 80 times more inhibitory than HS (mean difference of 1.9 logn0 in inhibitory titres).
Discussion
Most of the available methods for measuring allergenicity (reviewed by Stanworth, 1973) have not proved ideal for the purpose. For instance, measurements of weal and flare reactions in the skin of sensitised individuals are subject to numerous variables and consequently difficult to interpret in both man (Coca and Grove, 1925; Becker and Rappaport, 1948; Rappaport and Becker, 1949; Swain and Becker, 1952; Stanworth and Kuhns, 1965) and cattle (Black and Pay, 1975). The in vitro release of pharmacodynamic mediators from sensitised tissues (Lichtenstein and Osler, 1966; Lichtenstein et al., 1966) calls for highly specialised laboratory procedures, while radioimmunoassay methods (Wide et al., 1967) require anti-IgE sera which are not generally available for species other than man. The results obtained here suggest that the PCA-I test is a potentially useful method for demonstrating allergenicity which allows the activity of several sub-
253
stances to be titrated simultaneously in the skin of the same animal. This renders the assessments comparable and in the present case we were able to quantify the relative allergenicity of the two substances. In the past, PCA test variations have been observed which could be attributed to the sensitivity of the individual's skin employed (Beadle, 1971; Beadle and Pay, 1975; Black and Pay, 1975; Gershwin, 1981). In the experiments reported here, there was a 2-4-fold difference between the sensitivities of the goats, as indicated by the limiting dilutions of the antiserum injected with admixture of saline only (see Fig. 2). Likewise there was some animal to animal variation in the relationship between the inhibitory activity of BHK cell lysate and HS, and the difference between the mean titres was 2.1 and 1.6 log~0 on goats NT3 and NT4 respectively (see Fig. 3). Although these differences may be regarded as marginal, it seems desirable to repeat PCA and PCA-I tests routinely on several animals, as has previously been the practice (Black, 1977b). A quantitative relationship was demonstrated between the concentrations of the reacting substances and the titration endpoints were dependent on the dilutions of both the inhibitors and the antiserum employed. An idea of the sensitivity of the PCA-I test can be obtained from the protein concentration of the lysate at the titration endpoint. It can be calculated that the 1/2048 dilution which caused inhibition on goat NT4 contained approximately 1/zg of protein per 0.1 ml. In the case of purified allergen, however, the test would probably be capable of demonstrating concentrations lower than this.
Acknowledgements We wish to thank Mrs. E. Pink for excellent technical assistance and Mr. B. Dyson, Mr. N. Bedding and Mr. D. Saven for help with the animals.
References Beadle, G.G., 1971, M.V.Sc. Thesis, Liverpool University. Beadle, G.G. and T.W.F. Pay, 1975, Res. Vet. Sci.'19, 1. Becker, E.L. and B.Z. Rappaport, 1948, J. Allergy 19, 317. Black, L., 1977a, J. Immunol. Methods 15, 193. Black, L., 1977b, Vet. Rec. 100, 195. Black, L. and T.W.F. Pay, 1975, J. Hyg. Camb. 74, 169. Black, L., F.J.R.R. Menard, G.G. Beadle and T.W.F. Pay, 1975, J. Hyg. Camb. 75, 79. Coca, A.F. and E.F. Grove, 1925, J. Immunol. 10, 445. Gershwin, L.J., 1981, Amer. J. Vet. Res. 42, 1184. Levine, P. and A.F. Coca, 1926, J. Immunol. 11, 411. Lichtenstein, L.M. and A.G. Osler, 1966, J. Immunol. 96, 169. Lichtenstein. L.M., T.P. King and A.G. Osler, 1966, J. Allergy 38, 174. Patterson, R. and J. Correa, 1959, Int. Arch. Allergy 15, 335. Rappaport, B.Z. and E.L. Becker, 1949, J. Allergy 20, 358. Stanworth, D.R., 1959, Immunology 2, 384.
254 Stanworth, D.R., 1973, in: Frontiers of Biology: Immediate Hypersensitivity, eds. A. Neuberger and E.L Tatum (North-Holland Publishing Co., Amsterdam) pp. 127-179. Stanworth, D.R. and W.J. Kuhns, 1965, Immunology 8, 323. Stoker, M.G.P. and I. MacPherson, 1964, Nature (London) 203, 1355. Swain, H.H. and E.L. Becker, 1952, J. Allergy 23, 441. Wide, L., H. Bennich and S.G.O. Johannson, 1967, Lancet ii, 1105.
249
Elsevier JIM02816
Titration of Allergenicity using a Passive Cutaneous Anaphylaxis Inhibition Test L. B l a c k a n d M . J . F r a n c i s The Wellcome Foot and Mouth Disease Vaccine Laboratory, Pirbright, Woking, Surrey, U.K.
(Received 1 February 1983, accepted 25 May 1983)
A method for titrating allergenicity is described and validated by demonstrating that both baby hamster kidney cell lysate and hamster serum are capable of inhibiting the passive cutaneous anaphylaxis reactions produced by anti-BHK bovine serum in the skin of goats. Key words: cattle - reagins - passive cutaneous anaphylaxis - allergens
Introduction
Radioallergosorbent techniques (Wide et al., 1967) are commonly used to quantify substances which are allergenic for man. These methods, however, call for the use of specific anti-IgE sera which are seldom available for non-human species. In the past we used the passive cutaneous anaphylaxis (PCA) method to identify substances which were potentially allergenic for cattle (Black et al., 1975). This test, carried out by injecting cattle antiserum intradermally into goats, and injecting Evans blue and the suspect allergens intravenously 3 days later, was, however, not ideal for the quantitative assessment of allergenic activity. There have been reports that Prausnitz-Ktistner reactions in man could be inhibited by admixture of the antiserum with its specific allergen (Levine and Coca, 1926; Patterson and Correa, 1959) and this suggested that a method involving inhibition of PCA reactions might be useful for titrating the activity of allergens when the use of radioallergosorbent methods is precluded. In this study we aimed to validate the passive cutaneous anaphylaxis inhibition (PCA-I) method by measuring the inhibiting activity of baby hamster kidney (BHK) cell lysate on the action of its specific bovine-derived antiserum. We sought to demonstrate that inhibition endpoints are dependent on a quantitative interrelationship between the concentrations of the antiserum and the inhibitor. BHK cell lysate was selected as the antigen because previous results have demonstrated its ability to stimulate reaginic antibodies in cattle (Black, 1977a, b). We also took the opportunity of measuring the inhibition produced by dilutions 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
250 of hamster serum (HS) because allergenic cross-reactions between horse serum and epithelial cell derivatives have been previously demonstrated in the case of horse dander allergy in man (Stanworth, 1959).
Materials and Methods Animals Two white female Saanen goats about 10 months old were used. B H K cell lysate (Stoker and McPherson, 1964) Four litres of cell harvest containing 4.1 x 10 6 BHK-21 suspension culture cells/ml were centrifuged at 500 × g for 10 min and the sedimented cells were washed three times in phosphate buffered saline. The cells were then suspended in 100 ml distilled water and frozen and thawed three times by immersing in liquid nitrogen. The gross cell debris was sedimented at 1000 × g for 10 min and the supernatant clarified by spinning at 75,000 × g for 60 min. The concentration of protein in the supernatant, assessed by Bio-Rad Reagent (Bio-Rad Laboratories), was 23.6 m g / m l . The P C A - I test The goats were sedated by injecting 0.4 ml of 2% xylazine (Rompun, Bayer) intramuscularly and the hair over the chest walls and flanks was clipped. The skin on each side was marked out in a grid of 6 × 10 blocks, each about 3 cm × 4 cm. Bovine serum (No. 2358), which was known to contain anti-BHK reaginic antibodies, was made up in a doubling dilution series from 1 / 4 to 1/128 in normal saline and mixed with equal volumes of either B H K cell lysate (doubling dilutions from 1 / 8 to 1/2048) or HS (doubling dilutions from 1/1 to 1/256). (All subsequent dilutions are expressed as final dilutions after mixing.) The mixtures were left at 4°C for 24 h and then injected into the skin of the goats, 0.1 ml per block, using 1 ml plastic syringes and 26-gauge needles. The injections were administered in chequer board fashion, with the dilutions of antiserum running vertically and the dilutions of lysate or HS running horizontally (see Fig. 2). The antiserum-BHK cell lysate mixtures were injected on the left side of each animal and the antiserum-HS mixtures on the right. On each side a control series was also included comprising an antiserum dilution series mixed with an equal volume of saline only. Identical tests were performed on each of the goats to examine the reproducibility of the results. Three days later, 10 ml of 2% Evans blue dye followed by 6 ml of the B H K cell lysate were injected intravenously and, after 45 rain, the blue stains which appeared at the positive reaction sites were noted and photographed. Results
Fig. 1 is a photograph of the reactions on the right side of goat NT4 and Fig. 2 a - d represents diagrammatically all the reactions which were observed.
251
Fig. 1. The right side of goat NT4 showing the inhibitory activity of hamster serum on anti-BHK reaginic serum. For detailed annotation see Fig. 2d.
a). NT3,LHS: BHK
b~ NT4,LHS: lysate
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I I I-1-1~1 I I I I I I ! ! ! ! ~ I I I I 1 I
BHK
81
161 321 641 1281 2561
lysate
I I I
I I I I I
I I I I I
I I I I I
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~C~O00 DO C~ C~ n
3
c~ NT3,RHS:
d~ NT4,RHS:
HS
HS
Q. C ."*" ~'..
81 1*1.1.1.1-1.1.1-1.1 321 11161Ii Iolololo I I I ° °, °° ,• 6411 i I I I I I I ! I 1281 I I I I I I I I I I
81 1~32 I 64
l*l*l*l*l*l.l*l,l II I°1°1°1°1°1°1-1 II II II°l°lolel°l I I I I i I°l°l°l III]
2561
2561
I I I I I 1 I I i
I I I I I I I I I I
dilutions of i n h i b i t o r s - - ~ Fig. 2. The left and right sides of goats NT3 and NT4. The reaginic serum (dilutions from top to bottom) were mixed with equal volumes of either B H K cell lysate or hamster serum (HS) (dilutions from left to right). The black dots in the blocks represent reactions. The column at the extreme right of each box contains dilutions of the reaginic antiserum mixed with saline only. (Dilutions expressed as final dilution reciprocals.)
252
I
b) Goat No.
a) Goat No. NT 3
A O v-3.0
/SBHK
m/m
ly.~.
3.0
v
1.0
I
w/8
/
BHK lys.
,.o
2.0
.1¢
NT4
/,
0 0.9
1.0
1.2
1.5
1.8
0
0.9
1.2
1.5
dilution of reaginic antiserum(log10)
1.8
--
Fig. 3. The inhibiting titres of BHK cell lysate (BHK lys.) and hamster serum (H. Ser.) on dilutions of anti-BHK reaginic serum.
Both BHK cell lysate and HS inhibited the PCA reactions and the inhibition endpoints were dependent on the dilution of the reaginic antiserum employed. For instance Fig. 2d shows that the inhibiting titre of HS on goat NT4 was 1/4, 1/8, 1/32 and 1/128 when mixed with the reaginic antiserum diluted 1/8, 1/16, 1/32 and 1/64 respectively. When reaginic antiserum dilutions were mixed with saline only, the reactions occurred to a limiting dilution of 1/32 on goat NT3 and 1/64 on goat NT4 (see Fig. 2, column at the extreme right of each grid), reflecting the different sensitivities of the animals involved. In Fig. 3a and b the inhibition produced by the BHK cell lysate and HS have been plotted against the antiserum dilution and it is evident that the BHK cell lysate was approximately 80 times more inhibitory than HS (mean difference of 1.9 logn0 in inhibitory titres).
Discussion
Most of the available methods for measuring allergenicity (reviewed by Stanworth, 1973) have not proved ideal for the purpose. For instance, measurements of weal and flare reactions in the skin of sensitised individuals are subject to numerous variables and consequently difficult to interpret in both man (Coca and Grove, 1925; Becker and Rappaport, 1948; Rappaport and Becker, 1949; Swain and Becker, 1952; Stanworth and Kuhns, 1965) and cattle (Black and Pay, 1975). The in vitro release of pharmacodynamic mediators from sensitised tissues (Lichtenstein and Osler, 1966; Lichtenstein et al., 1966) calls for highly specialised laboratory procedures, while radioimmunoassay methods (Wide et al., 1967) require anti-IgE sera which are not generally available for species other than man. The results obtained here suggest that the PCA-I test is a potentially useful method for demonstrating allergenicity which allows the activity of several sub-
253
stances to be titrated simultaneously in the skin of the same animal. This renders the assessments comparable and in the present case we were able to quantify the relative allergenicity of the two substances. In the past, PCA test variations have been observed which could be attributed to the sensitivity of the individual's skin employed (Beadle, 1971; Beadle and Pay, 1975; Black and Pay, 1975; Gershwin, 1981). In the experiments reported here, there was a 2-4-fold difference between the sensitivities of the goats, as indicated by the limiting dilutions of the antiserum injected with admixture of saline only (see Fig. 2). Likewise there was some animal to animal variation in the relationship between the inhibitory activity of BHK cell lysate and HS, and the difference between the mean titres was 2.1 and 1.6 log~0 on goats NT3 and NT4 respectively (see Fig. 3). Although these differences may be regarded as marginal, it seems desirable to repeat PCA and PCA-I tests routinely on several animals, as has previously been the practice (Black, 1977b). A quantitative relationship was demonstrated between the concentrations of the reacting substances and the titration endpoints were dependent on the dilutions of both the inhibitors and the antiserum employed. An idea of the sensitivity of the PCA-I test can be obtained from the protein concentration of the lysate at the titration endpoint. It can be calculated that the 1/2048 dilution which caused inhibition on goat NT4 contained approximately 1/zg of protein per 0.1 ml. In the case of purified allergen, however, the test would probably be capable of demonstrating concentrations lower than this.
Acknowledgements We wish to thank Mrs. E. Pink for excellent technical assistance and Mr. B. Dyson, Mr. N. Bedding and Mr. D. Saven for help with the animals.
References Beadle, G.G., 1971, M.V.Sc. Thesis, Liverpool University. Beadle, G.G. and T.W.F. Pay, 1975, Res. Vet. Sci.'19, 1. Becker, E.L. and B.Z. Rappaport, 1948, J. Allergy 19, 317. Black, L., 1977a, J. Immunol. Methods 15, 193. Black, L., 1977b, Vet. Rec. 100, 195. Black, L. and T.W.F. Pay, 1975, J. Hyg. Camb. 74, 169. Black, L., F.J.R.R. Menard, G.G. Beadle and T.W.F. Pay, 1975, J. Hyg. Camb. 75, 79. Coca, A.F. and E.F. Grove, 1925, J. Immunol. 10, 445. Gershwin, L.J., 1981, Amer. J. Vet. Res. 42, 1184. Levine, P. and A.F. Coca, 1926, J. Immunol. 11, 411. Lichtenstein, L.M. and A.G. Osler, 1966, J. Immunol. 96, 169. Lichtenstein. L.M., T.P. King and A.G. Osler, 1966, J. Allergy 38, 174. Patterson, R. and J. Correa, 1959, Int. Arch. Allergy 15, 335. Rappaport, B.Z. and E.L. Becker, 1949, J. Allergy 20, 358. Stanworth, D.R., 1959, Immunology 2, 384.
254 Stanworth, D.R., 1973, in: Frontiers of Biology: Immediate Hypersensitivity, eds. A. Neuberger and E.L Tatum (North-Holland Publishing Co., Amsterdam) pp. 127-179. Stanworth, D.R. and W.J. Kuhns, 1965, Immunology 8, 323. Stoker, M.G.P. and I. MacPherson, 1964, Nature (London) 203, 1355. Swain, H.H. and E.L. Becker, 1952, J. Allergy 23, 441. Wide, L., H. Bennich and S.G.O. Johannson, 1967, Lancet ii, 1105.