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Tears and aqueous humor from horses inoculated with Leptospira contain antibodies which bind to cornea
Veterinary Immunology and Immunopathology, 14 (1987) 181-185 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
181
TEARS AND AQUEOUSHUMORFROMHORSES INOCULATEDWITH LEPTOSPIRA CONTAIN ANTIBODIES WHICH BIND TO CORNEA A.E. PAPJ~A, A.S. FERNANDEZ, C.G. SANTISTEBAN, R.A. BOWDENand S.I. CERONE Laboratorio de Inmunoqu~mica, Facultad de Cs. Veterinarias, Universidad Nacional del Centro, Pinto 399 (7000) Tandil (Argentina) (Accepted 9 September 1986) ABSTRACT Parma, A.E., Fernandez, A.S., Santisteban, C.G., Bowden, R.A. and Cerone, S . I . , 1987. Tears and aqueous humor from horses inoculated with Leptospira contain antibodies which b~n~ to cornea. Vet. Immunol. Immunopathol, 14: 181-185. An antigenic relationship between Leptospira interrogans and equine cornea was previously described by us. An enzyme-linked immunosorbent assay was employed in the present work to investigate the existence of anti-leptospira and anti-cornea antibodies in tears, aqueous humor and serum from horses inoculated i.m. with those antigens. Ten days after a booster by the same route, antibodies that bind to microtiter plates, coated with an homogenate of either equine cornea or leptospira, were detected in those fluids and in the sera. At the same time, the corneas of the horses began to develop a diffuse opacity. This finding of anti-leptospira antibodies in equine tears and aqueous humor shows the pathway along which they arrive at the cornea and bind to i t . INTRODUCTION Horses infected by Leptospira present several clinical disorders, one of them being recurrent i r i d o c y c l i t i s or periodic ophtalmia which alters the composition of the aqueous humor and impedes the nutrition of the ocular structures (Blogg and Coles, 1970), leaving sequelae such as i r i s atrophy, synechiae and corneal opacity (Jones, 1942; Heusser, 1948, 1952; Gamble et a l . , 1970a,b; Matthews and Handscombe, 1983). In a previous paper we demostrated an antigenic relationship between Leptospira and equine cornea (Parma et a l . , 1985). Horses inoculated i.m. with either cornea or different serovars of killed Leptospira interrogans developed corneal opacity. They produced antibodies which were isolated and purified, showing that they corresponded to the IgGb subclass. They bound to equine cornea in vivo and in v i t r o , as shown by immunofluorescence. As the cornea is an avascular tissue which is in contact with both aqueous humor and tears, we investigated the presence of anti-leptospira antibodies in those fluids of inoculated horses to explain the arrival of the antibodies at the cornea.
0165-2427/87/$03.50
© 1987 Elsevier Science Publishers B.V.
182
MATERIAL AND METHODS Animals and immunizations Cross bred horses were used. They were found to be negative f o r leptospiral antibodies and corneal opacity p r i o r to inoculation. Two horses were injected (i.m.) with 5 ml of an inoculum consisting of equal parts of Freund's incomplete adjuvant and 6 x 108 Leptospira c e l l s ml - I . This suspension was obtained as follows: Leptospira interro~ans serovars w o l f f i , tarassovi, pomona, icterohaemorrha~iae and hardjo (INTA-Castelar; Inst. Sanidad Ganadera) were harvested from Stuart's culture medium, washed with PBS, formol k i l l e d , suspended (6 x 108 bacteria ml - I ) in the same buffer and pooled in equal quantities. Another horse was inoculated (i.m.) with 2.5 ml of homogeneized normal equine cornea (5.7 mg of protein m l - l ) , obtained as previously indicated (Parma et a l . , 1985), mixed with an equal volume of Freund's incomplete adjuvant. After 24 days the inoculated animals received an i.m. booster with the respective antigens. Collection of samples Ten days a f t e r the booster, opthalmologic observations were carried out before the c o l l e c t i o n of samples. Tear samples were collected by using cellulose sponges which were inserted into the ventronasal conjunctival cul-de-sac and removed when throughly moistened with tears (2 to 3 min.). Tears were eluted by placing the sponges in 2 ml of saline solution for 24 h at 4°C (Glaze et a l . , 1984). For the c o l l e c t i o n of aqueous humor, horses were sedated with promazine chlorhydrate (Esparinal-Wyeth) and sodium pentothal (Abbot) and placed in l a t e r a l recumbency. A s t e r i l e 25-gauge needle and a i ml tuberculine syringe were used to withdraw 500 ul of f l u i d ; the needle was inserted into the anterior chamber of the eye through the l a t e r a l limbus (Glaze et a l . , 1984). Serum samples were simultaneously obtained. Fifteen days l a t e r , the ophthalmological examinations were repeated. Enzxme-linked immunosorbent assaX Ninety-six well m i c r o t i t e r plates (Linbro) were coated overnight with 50 ul of a 1:4 d i l u t i o n of a homogenate of either Leptospira (3.0 mg of protein m1-1) or equine cornea (5.7 mg of protein ml - I ) (Parma et a l . , 1985) in carbonate buffer (0.015 M Na2CO3- 0.035 M NaHCO3, pH 9.6). Plates were then washed 5 times with phosphate buffered saline (PBS) containing 0.10% Tween 20 and incubated overnight with PBS-Tween 20-0.5% bovine serum albumin. As the f i r s t antibody, 50 ul of the samples of serum (1:10 d i l u t i o n ) , tears (1:4 d i l u t i o n ) or aqueous humor (1:4 d i l u t i o n ) of each horse were used. After an incubation f o r 2 h at room temperature and a 4-step wash, 50 ul of an optimal d i l u t i o n of
183
rabbit anti-equine gammaglobulin serum (second antibody) were added and incubated for 2 h at room temperature. After a 4-step wash, 50 ul of a 1:200 dilution of goat anti-rabbit gammaglobulin conjugated to horseradish peroxidase (Sigma) were added and incubated in the conditions indicated above. After a final 4-step wash, 50 ul of 0.5 mg m1-1 benzidine and 0,003% H202 in PBS-Tween 20-bovine serum albumin were added to develop the colour (Tsang et a l . , 1983). The reaction was stopped with 0.5 M c i t r i c acid after 30 min at room temperature and the O.D. was measured by spectrophotometry (380 nm). Controls were included without antigen or f i r s t or second antibody. All assays were done in duplicate. RESULTS AND DISCUSSION Ten days after the horses received the booster, the ophthalmologic observations showed a diffuse corneal opacity. I t was most noticeable in the horse inoculated with equine cornea. The horse inoculated with cornea and one of those that had received Leptospira, developed a diffuse opacity in the lens. These findings confirm those in a previous paper (Parma et a l . , 1985). Fifteen days later, we observed a considerable increase of opacity in the lens of all horses. Meanwhile the opacity of the cornea was dispersing or had completely disappeared (Table I ) . One of the horses inoculated with Leptospira (C, Table I) showed considerable blindness in both eyes. TABLE I Ophthalmologic observations of horses injected with equine cornea and Leptospira Days post-booster Horse
Antigen injected
10 cornea
25 lens
cornea
lens
A
equine cornea
noticeable opacity
diffuse opacity
diffuse opacity
noticeable opacity
B
Leptospira
diffuse opacity
transparent
transparent
noticeable opacity
C
Leptospira
diffuse opacity
diffuse opacity
transparent
intense opacity
Table I I shows that the tears and aqueous humor of the horses inoculated with either Leptospira or equine cornea contain antibodies against the homologous antigen, and also present a cross-reaction against the other antigen Immunoglobulins of tears and aqueous humor from normal horses were unable to bind to microtiter plates coated with Leptos~ira and corneal antigens.
184
Controls performed without antigen or f i r s t
or second antibodies gave a
negative r e s u l t f o r ELISA. When the serum samples of these horses were assayed by ELISA they were strongly p o s i t i v e against Leptospira and equine cornea, showing, as occurred with tears and aqueous humor, an intense cross reaction (Table I I ) . This work was performed to f i n d out i f the a n t i - l e p t o s p i r a and anti-equine cornea antibodies were present in these f l u i d s . To do t h i s we chose the enzymelinked immunosorbent assay since i t is s e n s i t i v e enough to detect immunog l o b u l i n s in tears and aqueous humor. The r e s u l t s presented in the Table I I i n d i c a t e that the antigens employed to coat the m i c r o t i t e r plates (homogenate of Leptospira and equine cornea) are s a t i s f a c t o r y and could be recognized by t h e i r respective antibodies. TABLE I I Degree of p o s i t i v i t y of tears, aqueous humor and serum from horses inoculated with L. interrogans and cornea in ELISA against antigens of Leptospira and equine cornea Homogenized coating antigen Horse
Sample
Dilution
Leptospira
equine cornea
A
aqueous tears serum
1:4 1:4 1:10
+2 +2 +2
+2 +2 +2
B
aqueous tears serum
1:4 1:4 i:i0
+3 +4 +2
+i +i +l
C
aqueous tears serum
1:4 1:4 i:i0
+2 +2 +2
+i +i +2
D
aqueous tears serum
1:4 1:4 1:10
A: horse inoculated with equine cornea; B and C received L.interrogans; D: pool of samples from normal horses. Degree of p o s i v i t ~ : +1 (O.D. 380 nm: 0.200-0.400), +2 (O.D. 0.400-0.700), +3 (O.D. 0.700-1.100), +4 (O.D. over 1.100), negative ( - ) (O.D. under 0.200). The a n t i - l e p t o s p i r a and anti-equine cornea antibodies in tears and aqueous humor were detected at the moment when the corneal opacity developed in a l l horses. The opacity of the lens two weeks l a t e r would indicate that i t could also share some antigens with Leptospira. Studies are c u r r e n t l y being carried out in our laboratory to investigare t h i s hypothesis f u r t h e r .
185
The binding of antibodies to the corneal epithelium can have pathological consequences (Tizard, 1977). Our findings explain the pathway along wich the anti-leptospira and antiequine cornea antibodies arrive at the epithelium of the cornea, as well as the pathway by which these antibodies come into contact with the lens and, perhaps, bind to i t . ACKNOWLEDGEMENTS This work was supported by the Comisi6n de Investigaciones Cient(ficas Prov. Buenos Aires (grant 7044/84) and Consejo Nacional de Investig. Cient~ficas y T~cnicas (grants 1505 and 1611/84). The authors thank Eglez Montero for technical assistance. R.A. Bowden is a holder of a scholarship from the Comisi6n de Investigaciones Cient~ficas Prov. Buenos Aires. REFERENCES Blogg, J.R. and Coles, E.H., 1970. Aqueous Humor Proteins: A review. The Vet. Bulletin, 40: 347-351. Gamble, C.N., Aronson, S.B. and Brescia, F.B., 1970a. Experimental uveitis. The production of recurrent immunologic (Auer) uveitis and its relationship to increased uveal vascular permeability. Arch. Ophthalmol., 84: 321-330. Gamble, C.N., Aronson, S.B. and Brescia, F.B., 1970b. The pathogenesis of recurrent immunologic (Auer) uveitis. Arch. Ophthalmol., 84: 331-341. Glaze, M.B., McGuire, T.C., Schmidt, G.M. and Leid, R.W., 1984. Immunoglobulin levels in tears and aqueous humor of horses before and after diethylcarbamazine (DEC) therapy. Vet. Immunol. and Immunopathol., 7: 185198. Heusser, H., 1948. Die periodische Augenentzundung, eine Leptospirose? Schweiz.Arch. Tierheilk, 90: 287-314. Heusser, H., 1952. Zur Atiologie der periodischen Augenentzundung. Schweiz. Xrch. Tierheilk, 94: 296-306. Jones, T.C., 1942. Equine periodic ophthalmia. Am. J. Vet. Res., 3: 45-78. Matthews, A.G. and Handscombe, M.C., 1983. Uveitis in the horse: A review of the aetiological and immunopathological aspects of the disease. In: Equine Ophthalmology (suppl.). Equine Vet. J., 2: 61-64. Parma, A.E., Santisteban, C.G., Villalba, J.S. and Bowden, R.A., 1985. Experimental demonstration of an antigenic relationship between Leptospira and equine cornea. Vet. Immunol. and Immunopathol., 10: 215-224. Tizard, I.R., 1977. Type I l l hypersensitivity: Pathological consequences of immune complex deposition. In: An Introduction to Veterinary Immunology. Philadelphia, W. B. Saunders Co., pp. 289-302. Tsang, V.C.W., Peralta, J.M. and Simons, A.R., 1983. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophresis, pp. 377-391. In: J.J. Langone and H. van Vunakis (ed), Methods in Enzymology, vol. 92, part. E. Academic Press Inc., New York.
181
TEARS AND AQUEOUSHUMORFROMHORSES INOCULATEDWITH LEPTOSPIRA CONTAIN ANTIBODIES WHICH BIND TO CORNEA A.E. PAPJ~A, A.S. FERNANDEZ, C.G. SANTISTEBAN, R.A. BOWDENand S.I. CERONE Laboratorio de Inmunoqu~mica, Facultad de Cs. Veterinarias, Universidad Nacional del Centro, Pinto 399 (7000) Tandil (Argentina) (Accepted 9 September 1986) ABSTRACT Parma, A.E., Fernandez, A.S., Santisteban, C.G., Bowden, R.A. and Cerone, S . I . , 1987. Tears and aqueous humor from horses inoculated with Leptospira contain antibodies which b~n~ to cornea. Vet. Immunol. Immunopathol, 14: 181-185. An antigenic relationship between Leptospira interrogans and equine cornea was previously described by us. An enzyme-linked immunosorbent assay was employed in the present work to investigate the existence of anti-leptospira and anti-cornea antibodies in tears, aqueous humor and serum from horses inoculated i.m. with those antigens. Ten days after a booster by the same route, antibodies that bind to microtiter plates, coated with an homogenate of either equine cornea or leptospira, were detected in those fluids and in the sera. At the same time, the corneas of the horses began to develop a diffuse opacity. This finding of anti-leptospira antibodies in equine tears and aqueous humor shows the pathway along which they arrive at the cornea and bind to i t . INTRODUCTION Horses infected by Leptospira present several clinical disorders, one of them being recurrent i r i d o c y c l i t i s or periodic ophtalmia which alters the composition of the aqueous humor and impedes the nutrition of the ocular structures (Blogg and Coles, 1970), leaving sequelae such as i r i s atrophy, synechiae and corneal opacity (Jones, 1942; Heusser, 1948, 1952; Gamble et a l . , 1970a,b; Matthews and Handscombe, 1983). In a previous paper we demostrated an antigenic relationship between Leptospira and equine cornea (Parma et a l . , 1985). Horses inoculated i.m. with either cornea or different serovars of killed Leptospira interrogans developed corneal opacity. They produced antibodies which were isolated and purified, showing that they corresponded to the IgGb subclass. They bound to equine cornea in vivo and in v i t r o , as shown by immunofluorescence. As the cornea is an avascular tissue which is in contact with both aqueous humor and tears, we investigated the presence of anti-leptospira antibodies in those fluids of inoculated horses to explain the arrival of the antibodies at the cornea.
0165-2427/87/$03.50
© 1987 Elsevier Science Publishers B.V.
182
MATERIAL AND METHODS Animals and immunizations Cross bred horses were used. They were found to be negative f o r leptospiral antibodies and corneal opacity p r i o r to inoculation. Two horses were injected (i.m.) with 5 ml of an inoculum consisting of equal parts of Freund's incomplete adjuvant and 6 x 108 Leptospira c e l l s ml - I . This suspension was obtained as follows: Leptospira interro~ans serovars w o l f f i , tarassovi, pomona, icterohaemorrha~iae and hardjo (INTA-Castelar; Inst. Sanidad Ganadera) were harvested from Stuart's culture medium, washed with PBS, formol k i l l e d , suspended (6 x 108 bacteria ml - I ) in the same buffer and pooled in equal quantities. Another horse was inoculated (i.m.) with 2.5 ml of homogeneized normal equine cornea (5.7 mg of protein m l - l ) , obtained as previously indicated (Parma et a l . , 1985), mixed with an equal volume of Freund's incomplete adjuvant. After 24 days the inoculated animals received an i.m. booster with the respective antigens. Collection of samples Ten days a f t e r the booster, opthalmologic observations were carried out before the c o l l e c t i o n of samples. Tear samples were collected by using cellulose sponges which were inserted into the ventronasal conjunctival cul-de-sac and removed when throughly moistened with tears (2 to 3 min.). Tears were eluted by placing the sponges in 2 ml of saline solution for 24 h at 4°C (Glaze et a l . , 1984). For the c o l l e c t i o n of aqueous humor, horses were sedated with promazine chlorhydrate (Esparinal-Wyeth) and sodium pentothal (Abbot) and placed in l a t e r a l recumbency. A s t e r i l e 25-gauge needle and a i ml tuberculine syringe were used to withdraw 500 ul of f l u i d ; the needle was inserted into the anterior chamber of the eye through the l a t e r a l limbus (Glaze et a l . , 1984). Serum samples were simultaneously obtained. Fifteen days l a t e r , the ophthalmological examinations were repeated. Enzxme-linked immunosorbent assaX Ninety-six well m i c r o t i t e r plates (Linbro) were coated overnight with 50 ul of a 1:4 d i l u t i o n of a homogenate of either Leptospira (3.0 mg of protein m1-1) or equine cornea (5.7 mg of protein ml - I ) (Parma et a l . , 1985) in carbonate buffer (0.015 M Na2CO3- 0.035 M NaHCO3, pH 9.6). Plates were then washed 5 times with phosphate buffered saline (PBS) containing 0.10% Tween 20 and incubated overnight with PBS-Tween 20-0.5% bovine serum albumin. As the f i r s t antibody, 50 ul of the samples of serum (1:10 d i l u t i o n ) , tears (1:4 d i l u t i o n ) or aqueous humor (1:4 d i l u t i o n ) of each horse were used. After an incubation f o r 2 h at room temperature and a 4-step wash, 50 ul of an optimal d i l u t i o n of
183
rabbit anti-equine gammaglobulin serum (second antibody) were added and incubated for 2 h at room temperature. After a 4-step wash, 50 ul of a 1:200 dilution of goat anti-rabbit gammaglobulin conjugated to horseradish peroxidase (Sigma) were added and incubated in the conditions indicated above. After a final 4-step wash, 50 ul of 0.5 mg m1-1 benzidine and 0,003% H202 in PBS-Tween 20-bovine serum albumin were added to develop the colour (Tsang et a l . , 1983). The reaction was stopped with 0.5 M c i t r i c acid after 30 min at room temperature and the O.D. was measured by spectrophotometry (380 nm). Controls were included without antigen or f i r s t or second antibody. All assays were done in duplicate. RESULTS AND DISCUSSION Ten days after the horses received the booster, the ophthalmologic observations showed a diffuse corneal opacity. I t was most noticeable in the horse inoculated with equine cornea. The horse inoculated with cornea and one of those that had received Leptospira, developed a diffuse opacity in the lens. These findings confirm those in a previous paper (Parma et a l . , 1985). Fifteen days later, we observed a considerable increase of opacity in the lens of all horses. Meanwhile the opacity of the cornea was dispersing or had completely disappeared (Table I ) . One of the horses inoculated with Leptospira (C, Table I) showed considerable blindness in both eyes. TABLE I Ophthalmologic observations of horses injected with equine cornea and Leptospira Days post-booster Horse
Antigen injected
10 cornea
25 lens
cornea
lens
A
equine cornea
noticeable opacity
diffuse opacity
diffuse opacity
noticeable opacity
B
Leptospira
diffuse opacity
transparent
transparent
noticeable opacity
C
Leptospira
diffuse opacity
diffuse opacity
transparent
intense opacity
Table I I shows that the tears and aqueous humor of the horses inoculated with either Leptospira or equine cornea contain antibodies against the homologous antigen, and also present a cross-reaction against the other antigen Immunoglobulins of tears and aqueous humor from normal horses were unable to bind to microtiter plates coated with Leptos~ira and corneal antigens.
184
Controls performed without antigen or f i r s t
or second antibodies gave a
negative r e s u l t f o r ELISA. When the serum samples of these horses were assayed by ELISA they were strongly p o s i t i v e against Leptospira and equine cornea, showing, as occurred with tears and aqueous humor, an intense cross reaction (Table I I ) . This work was performed to f i n d out i f the a n t i - l e p t o s p i r a and anti-equine cornea antibodies were present in these f l u i d s . To do t h i s we chose the enzymelinked immunosorbent assay since i t is s e n s i t i v e enough to detect immunog l o b u l i n s in tears and aqueous humor. The r e s u l t s presented in the Table I I i n d i c a t e that the antigens employed to coat the m i c r o t i t e r plates (homogenate of Leptospira and equine cornea) are s a t i s f a c t o r y and could be recognized by t h e i r respective antibodies. TABLE I I Degree of p o s i t i v i t y of tears, aqueous humor and serum from horses inoculated with L. interrogans and cornea in ELISA against antigens of Leptospira and equine cornea Homogenized coating antigen Horse
Sample
Dilution
Leptospira
equine cornea
A
aqueous tears serum
1:4 1:4 1:10
+2 +2 +2
+2 +2 +2
B
aqueous tears serum
1:4 1:4 i:i0
+3 +4 +2
+i +i +l
C
aqueous tears serum
1:4 1:4 i:i0
+2 +2 +2
+i +i +2
D
aqueous tears serum
1:4 1:4 1:10
A: horse inoculated with equine cornea; B and C received L.interrogans; D: pool of samples from normal horses. Degree of p o s i v i t ~ : +1 (O.D. 380 nm: 0.200-0.400), +2 (O.D. 0.400-0.700), +3 (O.D. 0.700-1.100), +4 (O.D. over 1.100), negative ( - ) (O.D. under 0.200). The a n t i - l e p t o s p i r a and anti-equine cornea antibodies in tears and aqueous humor were detected at the moment when the corneal opacity developed in a l l horses. The opacity of the lens two weeks l a t e r would indicate that i t could also share some antigens with Leptospira. Studies are c u r r e n t l y being carried out in our laboratory to investigare t h i s hypothesis f u r t h e r .
185
The binding of antibodies to the corneal epithelium can have pathological consequences (Tizard, 1977). Our findings explain the pathway along wich the anti-leptospira and antiequine cornea antibodies arrive at the epithelium of the cornea, as well as the pathway by which these antibodies come into contact with the lens and, perhaps, bind to i t . ACKNOWLEDGEMENTS This work was supported by the Comisi6n de Investigaciones Cient(ficas Prov. Buenos Aires (grant 7044/84) and Consejo Nacional de Investig. Cient~ficas y T~cnicas (grants 1505 and 1611/84). The authors thank Eglez Montero for technical assistance. R.A. Bowden is a holder of a scholarship from the Comisi6n de Investigaciones Cient~ficas Prov. Buenos Aires. REFERENCES Blogg, J.R. and Coles, E.H., 1970. Aqueous Humor Proteins: A review. The Vet. Bulletin, 40: 347-351. Gamble, C.N., Aronson, S.B. and Brescia, F.B., 1970a. Experimental uveitis. The production of recurrent immunologic (Auer) uveitis and its relationship to increased uveal vascular permeability. Arch. Ophthalmol., 84: 321-330. Gamble, C.N., Aronson, S.B. and Brescia, F.B., 1970b. The pathogenesis of recurrent immunologic (Auer) uveitis. Arch. Ophthalmol., 84: 331-341. Glaze, M.B., McGuire, T.C., Schmidt, G.M. and Leid, R.W., 1984. Immunoglobulin levels in tears and aqueous humor of horses before and after diethylcarbamazine (DEC) therapy. Vet. Immunol. and Immunopathol., 7: 185198. Heusser, H., 1948. Die periodische Augenentzundung, eine Leptospirose? Schweiz.Arch. Tierheilk, 90: 287-314. Heusser, H., 1952. Zur Atiologie der periodischen Augenentzundung. Schweiz. Xrch. Tierheilk, 94: 296-306. Jones, T.C., 1942. Equine periodic ophthalmia. Am. J. Vet. Res., 3: 45-78. Matthews, A.G. and Handscombe, M.C., 1983. Uveitis in the horse: A review of the aetiological and immunopathological aspects of the disease. In: Equine Ophthalmology (suppl.). Equine Vet. J., 2: 61-64. Parma, A.E., Santisteban, C.G., Villalba, J.S. and Bowden, R.A., 1985. Experimental demonstration of an antigenic relationship between Leptospira and equine cornea. Vet. Immunol. and Immunopathol., 10: 215-224. Tizard, I.R., 1977. Type I l l hypersensitivity: Pathological consequences of immune complex deposition. In: An Introduction to Veterinary Immunology. Philadelphia, W. B. Saunders Co., pp. 289-302. Tsang, V.C.W., Peralta, J.M. and Simons, A.R., 1983. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophresis, pp. 377-391. In: J.J. Langone and H. van Vunakis (ed), Methods in Enzymology, vol. 92, part. E. Academic Press Inc., New York.