Identification of Multiple Equine Infectious Anemia Antigens by Immunodiffusion Reactions W. A. Malmquist and C. S. Becvar*
ABSTRACT Equine infectious anemia (EIA) cell antigens prepared f-rom infected equine spleen, equine leukocyte cultures or a persistently infected equine dermis cell line contained at least two serologically reacting components. For convenience one component was designated as soluble antigen (SA) and the other as cellassociated antigen (CAA). The SA appeared as a single component when it was prepared from EIA virus precipitated from infectious tissue culture fluid with polyethylene glycol and ether treated but it was mixed with CAA when the source was infected cells. Cytolytic or mechanical disruption of infected cells appeared to accelerate the release of CAA. Reaction to each component could be identified in double and radial immunodiffusion tests by increasing the concentration of SA in a twocomponent antigenic mixture. The CAA component does not appear to affect the value of the immunodiffusion test as a diagnostic aid.
RESUME Cette experience visait a demontrer que, dans l'anemie infectieuse equine, les antigenes cellulaires prepares 'a partir de la rate de chevaux infectes, de cultures de leucocytes de cheval, ou d'une lignee de cellules dermiques de cheval infectee en permanence, contiennent au moins deux constituants capables de produire une reaction serologique. Pour plus de commodite, on donna 'a l'un de ces constituants le nom d'antigene soluble et 'a l'autre, celui d'antigene associe 'a la cellule. L'antigiene soluble apparut comme un constituant simple, *National Animal Disease Center, North Central Region, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 50010. Submitted April 28, 1975.
Vol. 39
-
October, 1975
lorsquon le prepara a l'aide d'une souche du virus de l'anemie infectieuse equine, precipitee i partir d'un surnageant virulent de culture cellulaire, grace au polyethylene glycol, et traitee 'a l'ether; si on avait par ailleurs recours ai des cellules virulentes, il faisait corps avec l'antigene associe a la cellule. L'eclatement cytolytique ou mecanique des cellules infect&es sembla accelerer le relachement de l'antigiene associe 'a la cellule. II s'avera possible d'identifier la reaction de chacun des deux constituants, 'a l'aide des epreuves d'immunodiffusion double et radiale, en augmentant la quantite d'antigene soluble dans un melange des deux constituants antigeniques. L'antigiene associe 'a la cellule ne sembla pas influencer la valeur de l'epreuve d'immunodiffusion qu'on utilise comme moyen de diagnostic.
INTRODUCTION The double immunodiffusion test for the diagnosis of eguine infectious anemia (EIA) reported by Coggins and Norcross in 1970 (2) has proved to be a convenient and reliable procedure. Originally the antigen was processed from infected spleen obtained from acutely sick horses (1, 2) but EIA virus propagated in equine leukocyte cultures and then purified has been used (5). The virus produces complete cytolysis of in vitro propagated equine leukocytes (3) and undoubtedly various cellular components are released during lysis. We might expect, therefore, that EIA antigen processed from infected spleen may differ only quantitatively in components also present in antigen derived from equine leukocytes. We have previously reported that EIA virus could be propagated in a persistently infected equine dermis cell line (ED) with411
out producing any cytopathic effect (4). The infected cells disrupted by sonication and the virus in the infectious supernatant fluid concentrated by precipitation with polyethylene glycol could be used as sources of antigen. We concluded that the origin of a soluble supernatant antigen (SA) was primarily from virions because it was produced from precipitates of infectious fluid only after treating them with ether. On the other hand, antigen consisting of sonicated infected ED cells (ED-CA) did not require prior ether treatment and therefore resembled antigens present in infected spleen or equine leukocyte cultures. The evidence presented here supports the suggestion in an earlier report by Nakajima et al (6) that some EIA antigens contain more than one serologic reacting component. We are reporting results that indicate EIA-infected cellular material consists of at least two specific antigenic components as identified by double and radial immunodiffusion.
Equine infectious anemia leukocyte antigen (EL - CA) containing CAA was prepared from the cell debris of infected cultures that had undergone complete cytolysis. The cell debris was sedimented by centrifugation, resuspended in an equal volume of the supernatant and subjected to freezing and thawing.
MATERIALS AND METHODS CELL CULTURES
Equine dermis cell cultures persistently infected with the Wyoming strain of EIA virus were propagated in Eagle's MEM with nonessential amino acids and 10% fetal calf serum (4). Subcultures in the ratio of 1:2 or 1:3 were prepared five to seven days after initial planting. Equine leukocytes from a single donoi were cultured according to the method described by Kobayashi and Kono (3). The leukocyte donor was proved free of equine herpes virus, type 2, by the inoculation of cell culture fluids and cells into rabbit kidney cell cultures.
Fig. 1. Double immunodiffusion with a line of identity between EIA antigens in outer wells and a reference serum (S). Antigens are: (1) commercial EIA antigen, spleer. derived, (2) minced EIA-infected spleen, (3) disrupted EIA-infected equine leukocytes, (4) sonicated EIA-infected ED cells and (5) concentrated ether treated supernatant from EIA-infected ED cell cultures. A spur formed between antigens in wells No. 4 and No. 5.
ANTIGEN
Antigens were prepared as described previously except for minor modifications (4). Spleen antigen (SP-CA) for demonstrating a cell-associated antigen (CAA) consisted of finely minced infected tissue that reacted distinctly in the double immunodiffusion test against a standard test serum. It was stored at -70°C between tests.
412
Fg. 2. Double immunodiffusion test with supernatant
derived EIA antigen in wells No. 1 and No. 3 and EIAinfected ED cell antigen in well No. 2. A spur formed on each side of well No. 2.
Can. J. comp. Med.
uniformly milky in appearance. Some preparations of ED-CA were treated with ether to make them comparable with SA. The SA was prepared from culture fluids harvested seven or eight days after infected ED cell cultures were planted. Virus was precipitated from the infectious fluids with 8°% polyethylene glycol (MW 6,000),
Fig. 3. The heavy precipitin line evident in Fig. 2 between well No. 2 and the serum well (S) became separated when concentrated supernatant EIA stock antigen was mixed with the EIA-infected ED cell antigen. Spurs became the extremities of the new precipitin line.
Fig. 4b. Same as Fig. 4a except that 100 X concentrated supernatant EIA stock antigen was mixed with EIAinfected ED cell antigen to produce a 25 X final concentration. A second precipitin ring appears to have formed through separation of the single line seen in Fig. 4a.
Fig. 4a. Radial immunodiffusion reaction with EIA-infected ED cell antigen. Noninfected ED cell suspension is in the adjacent control well. Only one precipitin ring was produced.
The ED-CA containing CAA was prepared from infected ED cells scraped from culture flasks five to seven days after planting. The cells were washed three times in borate buffer (pH 8.6), resuspended in equal volumes of the buffer solution and sonicated' until the suspension became 'Branson Instruments, Inc., Stamford, Connecticut.
Vol. 39 - October, 1975
Fig. 4c. Same as Figs. 4a and 4b except that 100 X concentrated supernatant EIA stock antigen was mixed with EIA-infected ED cell antigen to produce a 50 X final concentration. The outside precipitin ring enlarged and became further separated from an internal ring.
413
sedimented by centrifugation, resuspended in borate buffer (pH 8.6) to make 1% of the original volume and treated with an equal volume of ether to release the antigen. Ether was removed by aspiration and evaporation under partial vacuum. Stock SA equal to 1% of the original volume of supernatant was designated as 100 X concentration. ANTISERA
trated (Fig. 2). A second component apparently reinforced the precipitin reaction with SA as indicated by the heavy precipitin line but it did not follow the line of identity produced by SA. By increasing the concentration of one reactant through the addition of SA in the ED-CA we could separate the heavy precipitin line and thereby reveal the existence of two components, SA and CAA, in ED-CA (Fig. 3).
Antisera against EIA virus were obtained from four horses experimentally infected for at least one month or longer with the Wyoming strain of virus, from one horse chronically infected with a Kentucky strain and from one field case. IMMUNODIFFUSION TESTS
Procedures for conducting double and radial immunodiffusion tests have been described (4). In some experiments the 1% Noble's special agar' was prepared in borate buffer pH 6.4 instead of pH 8.6 without producing any detectable differences. The concentration of SA in a two component antigenic mixture was increased by adding 100 X stock SA to achieve the optimal level for identifying separate precipitin reactions. In some experiments to demonstrate the number of reacting components in a mixture a single well was used but in experiments to prove identity between components in different mixtures adjacent wells were prepared so that precipitin rings would overlap and some would merge (7).
Fig. Sa. Radial immunodiffusion test with 50 X concentrated supernatant EIA antigen in well No. 1 and EIA-infected ED cell antigen in well No. 2. An arc of a precipitin ring occurs in the area between the wells.
RESULTS Double immunodiffusion tests with antigens prepared from infected spleen, equine leukocytes, ED cells and SA produced what appeared to be a line of identity except that a spur formed between the wells containing ED-CA and SA (Fig. 1, wells no. 4 and no. 5). This reaction was isolated by placing SA in wells on each side of a well containing ED-CA antigen as illus2Dlfco Labs., Detroit, Michigan.
414
Fig. Sb. Same antigens as in Fig. Sa except that 100 X concentrated supernatant EIA stock antigen was mixed with an equal quantity of EIA-infected ED cell antigen for well No. 2. A line of identity includes both wells, but a smaller precipitin circle surrounds only well No. 2.
Can. J. comp. Med.
In an alternate procedure in which SA supernatant as well as from infected cells and ED-CA mixtures were used in vary- where it may be present in an unorganized ing concentrations we could separate the form. The origin of CAA is uncertain but precipitin lines due to SA and CAA anti- we can assume that it is not derived from gens by radial immunodiffusion patterns. virions or it is present in much lesser Because the diameter of one precipitin quantity there than in the infected cells. ring is dependent upon the concentration The CAA is most likely a product of the of SA (4), addition of this component infected cell and coincidentally similar to produced a larger circle whereas the circle SA in size and concentration and becomes due to reaction with CAA remained fairly separated from SA only because it is not constant (Figs. 4a, 4b, 4c). When SA was precipitated with polyethylene glycol. The introduced into separate wells adjacent to possibility that CAA is an artifact resultED-CA wells so that the precipitin rings ing from the manipulation of SA has not due to the SA antibody system overlapped been ruled out but we have been unable and merged, a line of identity could be to produce it from SA under various exshown. An arc of a second precipitin ring perimental conditions. No significance has been attached to attributed to CAA present in ED-CA was produced in the area between the antigen the presence or absence of CAA as it may influence the double immunodiffusion test wells (Figs. 5a, 5b). Results indicated that SA derived from in the diagnosis of EIA. Immunofluoresinfectious supernatant fluid and from in- cence, also specific for EIA, would not fected cells were identical but CAA was identify which antigen-antibody reaction not present in detectable amounts in in- is involved. fectious supernatant concentrated by the described procedure. When SP-CA or ELCA were substituted for ED-CA, reactions REFERENCES indicating the presence of CAA were produced although somewhat weaker. BOULANGER, P., G. L. BANNISTER and S. P. All six antisera used in the double im- 1. CARRIER. Equine infectious anemia: preparation munodiffusion test with ED-CA reacted of a liquid antigen extract for the agar-gel immiinocomplement-fixation tests. Can. J. diffusion and positively against the SA and CAA comcomp. Med. 36: 116-123. 1972. COGGINS, L. and N. S. NORCROSS. Immunodifponents but not against noninfected ED 2. fusion reaction in equine infectious anemia. Cornell cells. Preinfection sera from experimenVet. 60: 330-335. 1970. K. and Y. KONO. Propagation and tally infected horses did not react against 3. KOBAYASHI, titration of equine infectious anemia virus in horse leukocyte cultures. Natn. Inst. Anim. Hlth Qt.. any of the components. Tokyo 7: 8-20. 1967. 4. MALMQUIST, W. A.,
DISCUSSION Finding multiple reacting components in crude antigens used for immunodiffusion tests is not unusual (7). In many tests, components will be revealed by distinctly separated precipitin lines. However, with SA and CAA in the EIA antigen-antibody system, the precipitin line due to each of the components appears to coincide and form a single antigen-antibody reaction. Only by altering the concentration of one of the reactants in a mixture or by using a single component to form a line of identity with another in a mixture could we show the presence of two antigens. From the results obtained, SA is likely derived from virions in the infectious
Vol. 39- October, 1975
D. BARNETT and C. S. BECVAR. Production of equine infectious anemia antigen in a persistently infected cell line. Arch. ges. Virusforsch. 42: 361-370. 1973. 5. NAKAJIMA, H. and C. USHIMI. Detection of precipitating antibody in equine infectious anemia by concentrated virus anti-en. Natn. Inst. Anim. Hith Ot., Tokyo 12: 47-53. 1972. 6. NAKAJIMA, H., C. USHIMI, Y. FUKUINAGA and K. HIRASAWA. Preparation of equine infectious anemia virus antigen for immunodiffusion test. Ar-.h. ges. Virusforsch. 42: 339-345. 1973. 7. WILLIAMS, C. A. and M. W. CHASE. Methods in Immninology and Immunochemistry. Volume 3. pp. 220-221. New York: Academic Press, Inc. 1971.
ADDENDUM Since the above manuscript was submitted and accepted for publication it was called to our attention that a second EIA antigen was described earlier by B. Toma and P. Goret (Bull. Acad. vet. Fr. 47: 101115. 1974). We have confirmed that the antigen called "C" by Toma and Goret is identical to one component in our infected cell cultures.
415
ABSTRACT Equine infectious anemia (EIA) cell antigens prepared f-rom infected equine spleen, equine leukocyte cultures or a persistently infected equine dermis cell line contained at least two serologically reacting components. For convenience one component was designated as soluble antigen (SA) and the other as cellassociated antigen (CAA). The SA appeared as a single component when it was prepared from EIA virus precipitated from infectious tissue culture fluid with polyethylene glycol and ether treated but it was mixed with CAA when the source was infected cells. Cytolytic or mechanical disruption of infected cells appeared to accelerate the release of CAA. Reaction to each component could be identified in double and radial immunodiffusion tests by increasing the concentration of SA in a twocomponent antigenic mixture. The CAA component does not appear to affect the value of the immunodiffusion test as a diagnostic aid.
RESUME Cette experience visait a demontrer que, dans l'anemie infectieuse equine, les antigenes cellulaires prepares 'a partir de la rate de chevaux infectes, de cultures de leucocytes de cheval, ou d'une lignee de cellules dermiques de cheval infectee en permanence, contiennent au moins deux constituants capables de produire une reaction serologique. Pour plus de commodite, on donna 'a l'un de ces constituants le nom d'antigene soluble et 'a l'autre, celui d'antigene associe 'a la cellule. L'antigiene soluble apparut comme un constituant simple, *National Animal Disease Center, North Central Region, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 50010. Submitted April 28, 1975.
Vol. 39
-
October, 1975
lorsquon le prepara a l'aide d'une souche du virus de l'anemie infectieuse equine, precipitee i partir d'un surnageant virulent de culture cellulaire, grace au polyethylene glycol, et traitee 'a l'ether; si on avait par ailleurs recours ai des cellules virulentes, il faisait corps avec l'antigene associe a la cellule. L'eclatement cytolytique ou mecanique des cellules infect&es sembla accelerer le relachement de l'antigiene associe 'a la cellule. II s'avera possible d'identifier la reaction de chacun des deux constituants, 'a l'aide des epreuves d'immunodiffusion double et radiale, en augmentant la quantite d'antigene soluble dans un melange des deux constituants antigeniques. L'antigiene associe 'a la cellule ne sembla pas influencer la valeur de l'epreuve d'immunodiffusion qu'on utilise comme moyen de diagnostic.
INTRODUCTION The double immunodiffusion test for the diagnosis of eguine infectious anemia (EIA) reported by Coggins and Norcross in 1970 (2) has proved to be a convenient and reliable procedure. Originally the antigen was processed from infected spleen obtained from acutely sick horses (1, 2) but EIA virus propagated in equine leukocyte cultures and then purified has been used (5). The virus produces complete cytolysis of in vitro propagated equine leukocytes (3) and undoubtedly various cellular components are released during lysis. We might expect, therefore, that EIA antigen processed from infected spleen may differ only quantitatively in components also present in antigen derived from equine leukocytes. We have previously reported that EIA virus could be propagated in a persistently infected equine dermis cell line (ED) with411
out producing any cytopathic effect (4). The infected cells disrupted by sonication and the virus in the infectious supernatant fluid concentrated by precipitation with polyethylene glycol could be used as sources of antigen. We concluded that the origin of a soluble supernatant antigen (SA) was primarily from virions because it was produced from precipitates of infectious fluid only after treating them with ether. On the other hand, antigen consisting of sonicated infected ED cells (ED-CA) did not require prior ether treatment and therefore resembled antigens present in infected spleen or equine leukocyte cultures. The evidence presented here supports the suggestion in an earlier report by Nakajima et al (6) that some EIA antigens contain more than one serologic reacting component. We are reporting results that indicate EIA-infected cellular material consists of at least two specific antigenic components as identified by double and radial immunodiffusion.
Equine infectious anemia leukocyte antigen (EL - CA) containing CAA was prepared from the cell debris of infected cultures that had undergone complete cytolysis. The cell debris was sedimented by centrifugation, resuspended in an equal volume of the supernatant and subjected to freezing and thawing.
MATERIALS AND METHODS CELL CULTURES
Equine dermis cell cultures persistently infected with the Wyoming strain of EIA virus were propagated in Eagle's MEM with nonessential amino acids and 10% fetal calf serum (4). Subcultures in the ratio of 1:2 or 1:3 were prepared five to seven days after initial planting. Equine leukocytes from a single donoi were cultured according to the method described by Kobayashi and Kono (3). The leukocyte donor was proved free of equine herpes virus, type 2, by the inoculation of cell culture fluids and cells into rabbit kidney cell cultures.
Fig. 1. Double immunodiffusion with a line of identity between EIA antigens in outer wells and a reference serum (S). Antigens are: (1) commercial EIA antigen, spleer. derived, (2) minced EIA-infected spleen, (3) disrupted EIA-infected equine leukocytes, (4) sonicated EIA-infected ED cells and (5) concentrated ether treated supernatant from EIA-infected ED cell cultures. A spur formed between antigens in wells No. 4 and No. 5.
ANTIGEN
Antigens were prepared as described previously except for minor modifications (4). Spleen antigen (SP-CA) for demonstrating a cell-associated antigen (CAA) consisted of finely minced infected tissue that reacted distinctly in the double immunodiffusion test against a standard test serum. It was stored at -70°C between tests.
412
Fg. 2. Double immunodiffusion test with supernatant
derived EIA antigen in wells No. 1 and No. 3 and EIAinfected ED cell antigen in well No. 2. A spur formed on each side of well No. 2.
Can. J. comp. Med.
uniformly milky in appearance. Some preparations of ED-CA were treated with ether to make them comparable with SA. The SA was prepared from culture fluids harvested seven or eight days after infected ED cell cultures were planted. Virus was precipitated from the infectious fluids with 8°% polyethylene glycol (MW 6,000),
Fig. 3. The heavy precipitin line evident in Fig. 2 between well No. 2 and the serum well (S) became separated when concentrated supernatant EIA stock antigen was mixed with the EIA-infected ED cell antigen. Spurs became the extremities of the new precipitin line.
Fig. 4b. Same as Fig. 4a except that 100 X concentrated supernatant EIA stock antigen was mixed with EIAinfected ED cell antigen to produce a 25 X final concentration. A second precipitin ring appears to have formed through separation of the single line seen in Fig. 4a.
Fig. 4a. Radial immunodiffusion reaction with EIA-infected ED cell antigen. Noninfected ED cell suspension is in the adjacent control well. Only one precipitin ring was produced.
The ED-CA containing CAA was prepared from infected ED cells scraped from culture flasks five to seven days after planting. The cells were washed three times in borate buffer (pH 8.6), resuspended in equal volumes of the buffer solution and sonicated' until the suspension became 'Branson Instruments, Inc., Stamford, Connecticut.
Vol. 39 - October, 1975
Fig. 4c. Same as Figs. 4a and 4b except that 100 X concentrated supernatant EIA stock antigen was mixed with EIA-infected ED cell antigen to produce a 50 X final concentration. The outside precipitin ring enlarged and became further separated from an internal ring.
413
sedimented by centrifugation, resuspended in borate buffer (pH 8.6) to make 1% of the original volume and treated with an equal volume of ether to release the antigen. Ether was removed by aspiration and evaporation under partial vacuum. Stock SA equal to 1% of the original volume of supernatant was designated as 100 X concentration. ANTISERA
trated (Fig. 2). A second component apparently reinforced the precipitin reaction with SA as indicated by the heavy precipitin line but it did not follow the line of identity produced by SA. By increasing the concentration of one reactant through the addition of SA in the ED-CA we could separate the heavy precipitin line and thereby reveal the existence of two components, SA and CAA, in ED-CA (Fig. 3).
Antisera against EIA virus were obtained from four horses experimentally infected for at least one month or longer with the Wyoming strain of virus, from one horse chronically infected with a Kentucky strain and from one field case. IMMUNODIFFUSION TESTS
Procedures for conducting double and radial immunodiffusion tests have been described (4). In some experiments the 1% Noble's special agar' was prepared in borate buffer pH 6.4 instead of pH 8.6 without producing any detectable differences. The concentration of SA in a two component antigenic mixture was increased by adding 100 X stock SA to achieve the optimal level for identifying separate precipitin reactions. In some experiments to demonstrate the number of reacting components in a mixture a single well was used but in experiments to prove identity between components in different mixtures adjacent wells were prepared so that precipitin rings would overlap and some would merge (7).
Fig. Sa. Radial immunodiffusion test with 50 X concentrated supernatant EIA antigen in well No. 1 and EIA-infected ED cell antigen in well No. 2. An arc of a precipitin ring occurs in the area between the wells.
RESULTS Double immunodiffusion tests with antigens prepared from infected spleen, equine leukocytes, ED cells and SA produced what appeared to be a line of identity except that a spur formed between the wells containing ED-CA and SA (Fig. 1, wells no. 4 and no. 5). This reaction was isolated by placing SA in wells on each side of a well containing ED-CA antigen as illus2Dlfco Labs., Detroit, Michigan.
414
Fig. Sb. Same antigens as in Fig. Sa except that 100 X concentrated supernatant EIA stock antigen was mixed with an equal quantity of EIA-infected ED cell antigen for well No. 2. A line of identity includes both wells, but a smaller precipitin circle surrounds only well No. 2.
Can. J. comp. Med.
In an alternate procedure in which SA supernatant as well as from infected cells and ED-CA mixtures were used in vary- where it may be present in an unorganized ing concentrations we could separate the form. The origin of CAA is uncertain but precipitin lines due to SA and CAA anti- we can assume that it is not derived from gens by radial immunodiffusion patterns. virions or it is present in much lesser Because the diameter of one precipitin quantity there than in the infected cells. ring is dependent upon the concentration The CAA is most likely a product of the of SA (4), addition of this component infected cell and coincidentally similar to produced a larger circle whereas the circle SA in size and concentration and becomes due to reaction with CAA remained fairly separated from SA only because it is not constant (Figs. 4a, 4b, 4c). When SA was precipitated with polyethylene glycol. The introduced into separate wells adjacent to possibility that CAA is an artifact resultED-CA wells so that the precipitin rings ing from the manipulation of SA has not due to the SA antibody system overlapped been ruled out but we have been unable and merged, a line of identity could be to produce it from SA under various exshown. An arc of a second precipitin ring perimental conditions. No significance has been attached to attributed to CAA present in ED-CA was produced in the area between the antigen the presence or absence of CAA as it may influence the double immunodiffusion test wells (Figs. 5a, 5b). Results indicated that SA derived from in the diagnosis of EIA. Immunofluoresinfectious supernatant fluid and from in- cence, also specific for EIA, would not fected cells were identical but CAA was identify which antigen-antibody reaction not present in detectable amounts in in- is involved. fectious supernatant concentrated by the described procedure. When SP-CA or ELCA were substituted for ED-CA, reactions REFERENCES indicating the presence of CAA were produced although somewhat weaker. BOULANGER, P., G. L. BANNISTER and S. P. All six antisera used in the double im- 1. CARRIER. Equine infectious anemia: preparation munodiffusion test with ED-CA reacted of a liquid antigen extract for the agar-gel immiinocomplement-fixation tests. Can. J. diffusion and positively against the SA and CAA comcomp. Med. 36: 116-123. 1972. COGGINS, L. and N. S. NORCROSS. Immunodifponents but not against noninfected ED 2. fusion reaction in equine infectious anemia. Cornell cells. Preinfection sera from experimenVet. 60: 330-335. 1970. K. and Y. KONO. Propagation and tally infected horses did not react against 3. KOBAYASHI, titration of equine infectious anemia virus in horse leukocyte cultures. Natn. Inst. Anim. Hlth Qt.. any of the components. Tokyo 7: 8-20. 1967. 4. MALMQUIST, W. A.,
DISCUSSION Finding multiple reacting components in crude antigens used for immunodiffusion tests is not unusual (7). In many tests, components will be revealed by distinctly separated precipitin lines. However, with SA and CAA in the EIA antigen-antibody system, the precipitin line due to each of the components appears to coincide and form a single antigen-antibody reaction. Only by altering the concentration of one of the reactants in a mixture or by using a single component to form a line of identity with another in a mixture could we show the presence of two antigens. From the results obtained, SA is likely derived from virions in the infectious
Vol. 39- October, 1975
D. BARNETT and C. S. BECVAR. Production of equine infectious anemia antigen in a persistently infected cell line. Arch. ges. Virusforsch. 42: 361-370. 1973. 5. NAKAJIMA, H. and C. USHIMI. Detection of precipitating antibody in equine infectious anemia by concentrated virus anti-en. Natn. Inst. Anim. Hith Ot., Tokyo 12: 47-53. 1972. 6. NAKAJIMA, H., C. USHIMI, Y. FUKUINAGA and K. HIRASAWA. Preparation of equine infectious anemia virus antigen for immunodiffusion test. Ar-.h. ges. Virusforsch. 42: 339-345. 1973. 7. WILLIAMS, C. A. and M. W. CHASE. Methods in Immninology and Immunochemistry. Volume 3. pp. 220-221. New York: Academic Press, Inc. 1971.
ADDENDUM Since the above manuscript was submitted and accepted for publication it was called to our attention that a second EIA antigen was described earlier by B. Toma and P. Goret (Bull. Acad. vet. Fr. 47: 101115. 1974). We have confirmed that the antigen called "C" by Toma and Goret is identical to one component in our infected cell cultures.
415
