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A microplate enzyme-linked immunosorbent assay method was developed for the measurement of bovine immunoglobulin G antibody specific to the envelope.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1981, p. 46-48

Vol. 13, No. 1

0095-1137/81/010046-03$02.00/0

Microplate Enzyme-Linked Immunosorbent Assay for Bovine Leukemia Virus Antibody GIORGIO

POLI,`*

ANDREA BALSARI,' ALESSANDRA TONIOLO,' WILMA PONTI,' AND GIUSEPPE

VACIRCA2

Istituto di Microbiologia e Immunologia Veterinaria' and Istituto di Patologia Speciale e Clinica Medica Veterinaria,2 Università di Milano, Milan, Italy

A microplate enzyme-linked immunosorbent assay method was developed for the measurement of bovine immunoglobulin G antibody specific to the envelope antigen (glycoprotein 60) of bovine leukemia virus. The test was then performed on 440 serum samples from dairy cows belonging to herds in which bovine leukemia was suspected or which were leukemia free, and the results were compared with those obtained with the gel-diffusion technique.

Several immunological tests, such as the indirect immunofluorescence test (2), the Ouchterlony immunodiffusion technique (1, 5), the complement fixation test (6), the radioimmunoassay (4), and the early polykaryocytosis inhibition (3) are used at present for detection of antibodies against bovine leukemia virus (BLV). None of these appears to be fully adequate for mass-screening purposes insofar as experimental ease, promptness of response, analytical reliability, and practicabiity are concerned. We have recently proposed the use of counterimmunoelectrophoresis (8) to overcome some of the limitations of the agarose immunodiffusion technique which is, at present, the method of choice in screening herds for BLV. This paper reports our evaluation of the enzyme-linked immunosorbent assay (ELISA) which has been shown to be a promising serodiagnostic test for other infectious diseases (9). After developing a microplate ELISA technique to detect bovine immunoglobulin G antibody specific for the envelope glycoprotein 60 antigen of BLV, we performed the test first on 210 serum samples from leukemia-free herds (as indicated by previous serological and clinical surveys) to define the upper limit for the negative population; then, the ELISA was performed on 230 serum samples from dairy cows (3 to 5 years old) belonging to four different herds in which BLV was suspected. The same samples were tested by the Ouchterlony immunodiffusion technique, and results of the two tests were compared to determine the specificity and sensitivity of our ELISA method. MATERIALS AND METHODS Antigen. A purified preparation of the envelope glycoprotein 60 of BLV was supplied by Behringwerke, A. G., Marburg/Lahn, West Germany. A working so46

lution contained 45 ,g of protein per ml in 0.1 M NaHCO3, pH 9.4. The optimal concentration of antigen for the ELISA was determined by chess-board titration; twofold dilutions of the antigen were reacted with a 1:20 dilution of a pool of bovine sera strongly positive for BLV antibody by the double-diffusion technique. The dilution of the antigen giving the highest absorbance reading, indicating saturation of binding sites on the solid phase, was chosen for performing

the test. The working solution of the antigen did not react in the ELISA with sera strongly positive for bovine viral diarrhea antibody as detected by the serum neutralization technique. Sera. Bovine field serum and standard reference serum samples shown to be positive, weakly positive, or negative by the Ouchterlony double-diffusion technique were tested. The sera were diluted 1:10 in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% bovine serum albumin for examination by ELISA. ELISA reagent and procedure. (i) Coating of the solid phase. Wells of Microelisa plates (Dynatech Laboratories, Inc., Alexandria, Va.) were sensitized by adding 0.2 ml of the working dilution of antigen per well. After incubation at 37°C for 3 h, the wells were washed three times with a solution containing 0.05% Tween 20, 0.5% bovine serum albumin, and 0.15 M NaCl in deionized water (washing solution). (ài) Enzyme-labeled anti-bovine immunoglobulins. Horseradish peroxidase (Boehringer, Mannheim, West Germany) and rabbit immunoglobulin G fractions of anti-bovine immunoglobulin G, purified by diethylaminoethyl-cellulose chromatography, were coupled by the periodate method of Nakane and Kawaoi (7) which was slightly modified to obtain an approximate horseradish peroxidase/immunoglobulin G molar ratio of 2. The conjugate was purified by gelfiltration on Sephadex G-200 columns (Pharmacia, Sweden) and used in the assay at a concentration of approximately 4 gg/mil in the same diluent used for the test sera. (iii) Substrate for the enzymatic reaction. oPhenylenediamine dihydrochloride (2 mg) was added to 10 ml of 0.25 M phosphate-citrate buffer containing

ELISA FOR BOVINE LEUKEMIA VIRUS ANTIBODY

VOL. 13, 1981

0.02% H202, to obtain a final pH of 4.8; this solution, kept in the dark, was used within 30 min. (iv) Procedure. The following scheme was used. To two wells of the microplate, coated with the antigen as described, was added 0.2 ml of a 1:10 dilution of each test serum. After 2 h of incubation at 37°C, the wells were washed three times with 0.35 ml of the washing solution per well. Then, 0.2 ml of the enzymelabeled anti-bovine immunoglobulin per well was added. After 30 min of incubation at 37°C, washing occurred as just described. Next, 50 pl of the substrate per well was added. After 5 min of incubation at room temperature, 50 gl of 2 N H2SO4 per well was added to stop the reaction. Absorbance at 492 nm was determined by Microelisa Reader AM 115 (Dynatech). Ouchterlony immunodiffusion technique. The procedure and the interpretation of the reactions were previously described (8). RESULTS

Establishment of the working dilution of test sera for the microplate ELISA. The ideal ELISA should provide a quantitative answer derived from a single reading at a determined dilution of serum rather than from a titration curve. Besides advantages in terms of experimental ease, this could circumvent problems related to titer evaluation (measurement at levels close to the assay sensitivity linit). We chose a working dilution of antigen at which both the high titer samples (bell-shaped curve) and the low titer samples (which influence the sensitivity and discriminating potency

A492 SINGLE REAOING

DILUTION IN NS O 100% PS * 207. PS a 10X PS

05-

-

16

256

I 4096

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--

DILUTION FACTOR

FIG. 1. Dilution curves of a positive sample (PS) increasingly diluted with a negative sample (NS) (immunoglobulin G ELISA). Optimal dilution range is indicated by dotted lines.

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TABLE 1. Classification of the samples (n = 440) by the immunodiffusion and the ELISA response (mU) Sample Immunodiffusion' classificaNo. tion

ELISAb No.

Mean + SD Range

415 583 ± 172 199 199 Positive aIdentity precipitation bands were classified as positive regardless of the type, intensity, or position of the band. b Optical density values; SD, standard deviation.

of the test) gave reliable results (Fig. 1). In practical terms, any dilution in the range of 1:10 to 1:100 seemed suitable. Definition of negative ELISA optical density value. A total of 210 serum samples from leukemia-free herds giving a negative reaction in the Ouchterlony immunodiffusion test were used to assess the normal range of ELISA values. In the ELISA system, this population gave a mean value and standard deviation (SD) of 136 ± 30 mU. Therefore, assuming a 1% probability of misclassification (i.e., i + 3 SD), the upper limit for the negative population is 224 mU. Analysis of the sample population from herds with suspected BLV. From an analysis of the ELISA data of a test population from herds with suspected bovine leukemia, 23 of the 230 serum samples gave negative results. The remaining 207 positive samples gave a mean value and an SD of 583 ± 172 mU in the ELISA system. This corresponds to a distribution ranging, at a 95% probability (i - 2 SD to i + 2 SD), from 237 to 927 mU, without any overlap with the previously defined negative class. A comparison of the results with those obtained by the reference immunodiffusion technique demonstrated that (i) no ELISA-negative samples were classified as positive by the reference method, and (ii) eight ELISA-positive samples were classified as negative by the doublediffusion technique. A further analysis of these eight samples gave an i and SD of 325 ± 45, with an experimental range from 254 to 380 mU and giving, at a 95% confidence limit, a range from 210 to 415 mU. Assuming that the discrepancies with respect to the reference method are related to improved sensitivity of the ELISA method, we have tentatively assumed, for the borderline population (i.e., weakly positive), the interval reported in Table 1. DISCUSSION conclusions Two emerge from these results: (i) the ELISA method is valid for the detection of the BLV antibody, showing substantial agree-

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J. CLIN. MICROBIOL.

POLI ET AL.

ment in terms of positive and negative samples with the immunodiffusion technique, and (ià) the data can be expressed as single readings which provide a quantitative answer. On the other

hand, the immunodiffusion technique (which is the most widely used for BLV serodiagnosis) gives an all-or-nothing response. Moreover, despite the small number of samples examined, it is possible to assume tentatively a range of values for a borderline population so that the samples can be clearly classified by the ELISA method as either negative, weakly positive, or

4.

5.

6.

positive. LITERATURE CITED 1. Baumgartner, L. E., C. Olson, J. M. Miller, and M. J. Van der Maaten. 1975. Survey for antibodies to leukemia (C-type) virus in cattle. J. Am. Med. Assoc. 166: 249-251. 2. Ferrer, J. F., L. Avila, and N. D. Stock. 1972. Serological detection of type C viruses found in bovine cultures. Cancer Res. 32:1864-1870. 3. Guillemain, B., R. Mamoun, D. Levy, T. Astier, K. Irgens, and A. L. Parodi. 1977. Early polykaryocytosis

7. 8.

9.

inhibition: a simple quantitative in vitro assay for the detection of bovine leukemia virus infection in cattle. Eur. J. Cancer 14:811-827. Levy, D., L. Deshayes, A. L. Parodi, J. P. Levy, J. R. Stephenson, S. G. Devare, and R. V. Gilden. 1977. Bovine leukemia virus specific antibodies among French cattle. II. Radioimmunoassay with the major structural protein (BLV p24). Int. J. Cancer 20:543550. Mammerickx, M., D. Portelle, R. Kettmann, J. Ghysdael, A. Burny, and D. Dekegel. 1976. Diagnostic test of bovine leukemia. Comparison between a hematological test and the serological diagnosis. Eur. J. Cancer 12:433-439. Miller, J. M., and M. J. Van der Maaten. 1974. A complement fixation test for the bovine leukemia (Ctype) virus. J. Natl. Cancer Inst. 53:1699-1702. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22:1084-1086. Poli, G., O. Pozza, W. Ponti, A. Balsari, and G. Vacirca. 1980. Application of counterimmunoelectrophoresis for a rapid serodiagnosis of enzootic bovine leukosis. Br. Vet. J. 136:51-55. Sever, J. L., and D. L. Madden. 1977. Enzyme linked immunosorbent assay (ELISA) for infectious agents. J. Infect. Dis. 136(Suppl.):S258-S340.