Br. vet.]. (1980). I36, 25I
APPLICATION OF COUNTERIMMUNOELECTROPHORESIS FOR A RAPID SERODIAGNOSIS OF ENZOOTIC BOVINE LEUKOSISt BY G. POL!, 0. POZZA, * W. PONTI, A. BALSARI AND G. VACIRCA*
lstituto di Microbiologia e lmmunologia Veterinaria and *Istituto di Patologia Speciale e Clinica Medica Velerinaria, Universita di Milano, Via Celona 10, 20133 Milano, Italy
SUMMARY
Counterimmunoelectrophoresis performed both on agarose slides and cellulose acetate strips allows rapid detection (2 to 4 h) of antibodies to the glycoprotein antigen of enzootic bovine leukosis virus. The method, tested on field sera from a leukosis-infected herd, detected the same reactors as the classic Ouchterlony double diffusion technique.
INTRODUCTION
One or more types of specific antibodies to either glycoproteins of the viral envelope, internal constituents of the viral particle or non-structural virus-induced protein can be detected in the sera of cattle naturally infected with enzootic bovine leukosis (EBL) virus. Four diverse methods are at present used in the serodiagnosis of EBL: indirect immunofluorescence (Gillette, Olson & Tekeli, 1969; Ferrer, Avila & Stock, 1972; Ferrer el al., 1974; Ressang el al., 1974), Ouchterlony's double diffusion technique (Miller & Olson, 1972; Olson el al., 1973; Baumgartener el al., 1975; Onuma et al., 1975; Mammerickx et al., 1976), complement fixation test (Miller & VanDer Maaten, 1974; Miller, VanDer Maaten & Gustafson, 1974), and the indirect immunoperoxidase test (Ressang el al., 1976). Of these, Ouchterlony's bidimensional immunodiffusion technique is usually the method of choice, because of its simplicity of execution, in screening herds for EBL ; however this method has its limitations in that its sensitivity is inferior in comparison with the other procedures(Ferrer et al., 1974; Ressanget al., 1976) and it is sometimes necessary to wait 48 to 72 h to obtain a definite reading. To overcome this apparent deficiency in the Ouchterlony technique, we have designed a test for the rapid serodiagnosis of EBL utilizing counterimmunoelectrophoresis, which is in itself a modification of the bidimensional immunodiffusion procedure. tCommunication given at the Conference on Bovine Leukosis in Milan, June 1979.
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y-globuline
1 e=:):=~ i precipitate
Fig. 1. Diagrammatic representation of countcrimmunoelectrophoresis .
Counterimmunoelectrophoresis, described for the first time by Kohn (1968), utilizes the Ouchterlony double diffusion technique, but is performed in an electric field . In such a system, the gammaglobulins move towards the cathode while the major part of the antigens, having a different electrophoretic motility, migrate towards the anode. A precipitate forms at the point of encounter of the two reagents (Fig. 1). In the following experiment, we utilized the counterimmunoelectrophoresis technique to detect specific antibodies in the sera of 400 cows belonging to a local dairy herd in which EBL was suspected. The same samples were tested by the Ouchterlony bidimensional gel technique to compare the sensitivity of the new procedure. MATERIALS AND METHODS
Antigens The antigen used in the experiment consisted of a purified preparation of EBL virus glycoprotein, obtained from an ovine cell-line showing persistent infection (antigen preparation furnished by Pitman-Moore, Inc. (USA), Behringwerke AG (Germany), Seromed (Monaco) through Gelman (Italy) and A .D.R.I. (Canada)). Sera Bovine field seta and standard reference sera, respectively positive, weakly positive or negative in the agarose gel diffusion technique were utlized in the experiment reference sera furnished by Pitman-Moore, Inc. (USA) and Behringwerke AG (Germany)).
· Counterimmunoelectrophoresis in agarose Preliminary tests were conducted with agarose at various concentrations in different buffer solutions (phosphate, borate and tris-glycine) with additions of NaCl or polyethyleneglycol (PEG 6000) in varying concentrations to allow the establishment of optimum conditions. Five ml of 0 · 8% agarose in a tris-glycine buffer solution (pH 9), with the addition of 0 · 85% NaCI, were layered on glass slides to form a layer 3 mm deep. Wells of 3 mm diameter and 2 mm apart were made as in Fig. 2. The slides were then placed on the bridge of the electrophoretic chamber and connected with the trisglycine buffer solution (pH 9) by strips of filter paper. The wells were filled with the reagents (25 ,ul) positioning the antigen towards the cathode while the test sera and the standard reference serum were positioned towards the anode. The electrophoresis was run at 200 V for 4 h.
s~
-
RS Ag
+
Se RS
Fig. 2 . Co unterimmun ockctrophorcsi s o n aga rosc . Ag EBL. viru s glyco protein , RS stand a rd rea gent serum . 5 1 . 5 2 and 5 1 sera under test .
RS
Ag
I
TS
Fig. 3. Countcrimmunoclectrophorcsis on cel lu lose acetate . AG EBL. ,·irus gl yco p ro tein . RS standard reagen t seru tn , TS scrun1 under test.
SERODIAGNOSIS OF ENZOOTIC BOVINE LEUKOSIS
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Counter-immunoelectrophoresis on cellulose acetate membrane Vergani's technique was used (Vergani, 1971): gelatinized cellulose acetate strips (Cellogel- Chemetron, Milan), previously buffered for 10 min in a tris -glycine solution (pH 9) containing 2% PEG 6000, were dried between two strips of filter paper to remove excess liquid and then stretched on the electrophoresis bridge. Ten ILl of antigen were deposited towards the cathode, 3 em from the border of the bridge; in front of every antigen deposit 10 ILl of test sera or standard reference serum were placed at a distance of 1 · 5 em towards the anode. Electrophoresis was run in the buffer solution already described by applying 200 V for 40 min. At the end of the migration, the strips were washed for 1 h in a 0 · 9% saline solution containing 2% PEG 6000, keeping them shaken and changing the solution every 15 min. The lines of precipitation on the strips can be observed by staining with Comassie brilliant blue following the technique of Pizzolato, Pizzolato & Agostoni (1972). In both the methods described above, a positive reaction is observed as an arc of precipitation between the antigen deposit and that of the test serum. Bidimensional immunodiffus-ion Ouchterlony's bidimensional double diffusion technique (Ouchterlony, 1948) was used . Six ml of medium (0 · 8% agarose in a tris buffer solution pH 7 · 2 with the addition of either 0 · 85 or 8 · 5% NaCl, depending on the antigen used) were poured into a Petri dish (5 · 5 em diam .) to form a layer of about 2 to 3 mm. In every quadrant of the dish a series of seven wells of 5 mm diameter and 2 mm apart were made, with six wells encircling a seventh. Seventy ILl of the antigen were deposited in the central well. The test sera were deposited in quantities of 70 ILl in three of the peripheral wells, which alternate between the other three wells containing the standard reference serum. The dishes were placed in a humid chamber at 25°C and the lines of precipitation read at 12, 24, 48 and 72 h. RESULTS
Counterimmunoelectrophoresis, conducted on agarose or on cellulose acetate membrane, proved extremely sensitive in detecting antibodies to glycoprotein antigens of EBL virus . With this method, precipitation lines can be observed after only 2 to 4 h (according to the procedure used) and therefore, in less time than by the Ouchterlony bidimensional immunodiffusion method. Using counterimmunoelectrophoresis on agarose and suitably arranging the sera, it is possible to observe , as in bidimensional immunodiffusion, a fusion of the bands of precipitation and thus to control the identity between the reactions of the test sera and those of the standard reference serum (Fig. 2) . In working with counterimmunoelectrophoresis on cellulose acetate membrane, because of the risk of mixing the reagents, the deposits could not be placed close enough to each other to produce the fusion of the arcs of precipitation whose specificity is, however, confirmed by their form and their position on the strips in respect to the precipitate of the standard reference serum (Fig. 3).
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In regard to investigations conducted on 400 bovine sera obtained from a local dairy herd suspected of EBL, we observed that with counterimmunoelectrophoresis the percentage of positive sera (82%) was greater than the number detected by the bidimensional immunodiffusion technique (about 78%). DISCUSSION
The results of trials conducted to evaluate the validity of counterimmunoelectrophoresis in the serodiagnosis of EBL demonstrate that this technique can substantially improve the sensitivity and reaction time observed in Ouchterlony's bidimensional immunodiffusion technique. Of the two supports used in the experiment (agarose and cellulose acetate), both appear suitable, but with some differences in procedure, in revealing specific antibodies to EBL virus glycoprotein. The use of agarose allows an easy detection of identity reactions between the precipitation lines of the positive sera under test and that of the standard reference serum. On the other hand, cellulose acetate membranes require only a microquantity of reagents (about 10 1£l); moreover, the stained precipitation arcs remain permanently visible when the strips are preserved in a 5% acetic acid solution. It is important to remember that counterimmunoelectrophoresis conducted on cellulose acetate membrane can be rendered even more sensitive by using an indirect immunofluorescent reaction (Poli et al., 1979): after the migration of the reagents, a fluorescent anti-bovine gammaglobulin serum is placed on the area of the membrane lying between the reagent deposits. After appropriate incubation and successive removal of the antiserum which remains unbound, the strips are then read by utilizing UV light which enables observation of the new antigen-antibody complex which will fluoresce. The immunofluorescence, besides substantially increasing the sensitivity of the test, also allows for identification of the class of immunoglobulin (IgM, IgG or IgA) involved in the reaction. REFERENCES
BAUMGARTENER, L. E. , OLSON, C., MILLER, J. M. & VANDER MAATEN , M. j. (1975).joumal of the American Veterinary Medical Association 166, 249. FERRER, j. F., AVILA, L. & STOCK, N.D. (1972) . Cancer Research 32, 1864. FERRER, j. F., ABT, D. A ., BHATT, D. M. & MARSHAK, R . R. (1974). Cancer Research 34, 893. GILLETTE, K. G., OLSON, C. & TEKEL!, S. (1969). Americanjournal of Veterinary Research 30, 975. KOHN, J. (1968). In Chromatographic and Electrophoretic Techniques, ed. I. Smith, London : Heinemann. Vol. II, p. 137. MAMMERICKX, M. , PORTETELLE, D., KETTMANN, R., GHYSDAEL, j., BURNY, A. & DEKEGEL, D. (1976) . European journal of Cancer I2, 433. MILLER, J. M. & OLSON, C. (1972).journal of the National Cancer Institute 49, 1459. MILLER, J. M. & VANDER MAATEN, M. J. (1974) . joumal of the National Cancer Institute 53, 1699. MILLER, J. M., VAN DER MAATEN, M. J. & GUSTAFSON, G. A. (1974). 17th Annual Proceedz'ngs of the American Association of Veten'nary Laboratory Diagnosticians, p . 207 . OLSO N, C . , Hoss, H . E., MILLER, j. M. & BAUMGARTENER, L. E. (1973). joumal of the American Veterinary Medical Association 162, 355.
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(Accepted for publication 7 November 1979)