Detection of Culture-Derived Babesia bovis Exoantigen Using a

Vol. 25, No. 9

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1987, p. 1648-1652 0095-1137/87/091648-05$02.00/0 Copyright C 1987, American Society for Microbiology

Detection of Culture-Derived Babesia bovis Exoantigen Using Two-Site Enzyme Immunoassay

a

FEDERICO MONTEALEGRE,'t SONIA MONTENEGRO-JAMES,"2 IBULAIMU KAKOMA,1* AND MIODRAG RISTIC' Department of Veterinary Pathobiology, College of Veterinary Medicine, University ofIllinois, Urbana, Illinois 61801,' and Instituto de Investigaciones Veterinarias, Maracay, Venezuela2 Received 30 January 1987/Accepted 1 June 1987

Soluble exoantigens in the supernatants of Babesia bovis cultures have been shown to be efficient immunogens against bovine babesiosis. We used a two-site enzyme immunoassay to monitor the release of these antigens during in vitro cultivation. Bovine immunoglobulin G was isolated from serum of an adult cow previously immunized with culture-derived B. bovis exoantigens and challenged via needle with virulent parasites. The specific immunoglobulin G was used as a capture antibody and as an enzyme-conjugated recognizing antibody. The optimal protein concentration of capture antibody was 10 ,ug/ml. The 24-h cultures showed the greatest antigen concentration. The test was sensitive for detection of differences in species-specific antigenic activity among B. bovis isolates, for determining loss of antigenicity during storage and formalinization, and for monitoring the kinetics of exoantigen release during in vitro cultivation. Antigens cross-reactive with the other major Babesia species of cattle, Babesia bigemina, were also detected with this assay. The high specificity, sensitivity, and reproducibility of this technique should facilitate detection and quantitation of Babesia antigens during purification and in standardization of candidate immunogens.

streptomycin, 100 U of penicillin per ml, released hemoglobin, and soluble parasite antigens, was centrifuged at 12,100 x g for 30 min at 4°C, filtered through a 0.45-,u.m-pore-size membrane filter (Millipore Corp., Bedford, Mass.), and either stored at -70°C or lyophilized and kept at 4°C. The reactivity of B. bovis exoantigens was also measured in pooled culture supernatant fluids stored as described above and treated with Formalin (0.1% final concentration) before storage. In addition, normal control supernatant from uninfected cultures was similarly prepared. Protein concentrations were determined by the method of Lowry et al. (10). At the Veterinary Research Institute in Maracay, Venezuela, the same procedure was followed to obtain exoantigens derived from in vitro cultures of B. bovis isolates from Venezuela, Argentina, and Ecuador. Antisera and conjugate. An adult Holstein cow was injected subcutaneously with 470 mg of lyophilized supernatant fluid containing B. bovis exoantigens from the Mexican strain, reconstituted in 1 ml of distilled water supplemented with 1 mg of Quil-A Saponin (Accurate Chemical & Scientific Corp., Westbury, N.Y.), on days 0 and 21. The cow was challenged via needle 112 days later with 108 B. bovisinfected erythrocytes. A serum antibody titer of 1:163,840 was observed on day 21 postchallenge, as measured by the indirect fluorescent antibody test (5). Immune IgG isolated by using caprylic acid (21) and further purified with DEAEcellulose (Whatman Inc., Clifton, N.J.) was conjugated to horseradish peroxidase (25) (type VI; Sigma Chemical Co., St. Louis, Mo.). At the Veterinary Research Institute, a conjugate using anti-B. bovis IgG (Venezuelan strain) was prepared as described above. Two-site immunoassay. A two-site immunoassay (3, 24) developed for the detection of culture-derived B. bovis exoantigens consists of four major steps: (i) binding purified anti-B. bovis IgG to microtiter wells, (ii) adding culture supernatant fluids containing soluble antigens, (iii) adding horseradish peroxidase-labeled anti-B. bovis IgG, and (iv) adding substrate and recording the subsequent amount of

The development of the continuous in vitro cultivation system for Babesia bovis by the microaerophilous stationary

phase techniques) (9) has facilitated several studies on this parasite and its products, e.g., exoantigens. Soluble exoantigens are defined as proteinaceous moieties present in culture supernatants and in the plasma of infected animals. James et al. (6) demonstrated three major soluble exoantigens in the supernatant fluid of B. bovis cultures that have been shown to induce protective immunity against bovine babesiosis (8, 14, 16, 17, 23). Reliable methods for detection and quantitation of soluble exoantigens have yet to be developed, and the use of enzyme immunoassays (EIAs) in bovine babesiosis has so far been limited to antibody detection (1, 2, 15, 26). In the present communication, we describe a two-site EIA using bovine immunoglobulin G (IgG) specific to B. bovis and demonstrate its potential for the quantitative assay of culture-derived B. bovis exoantigens and for monitoring antigenic stability under conditions commonly used for preservation and storage of various immunogens.

MATERIALS AND METHODS

Exoantigens. The B. bovis Mexican strain, described by Smith et al. (19), was used as a source of exoantigen. B. bovis parasites were cultured in 150-cm2 flasks by the method of Levy and Ristic (9). Cultures were incubated at 37°C under an atmosphere of 5% C02-air in 3-day cycles. The supernatant fluid was rernoved daily and replaced with fresh medium. The percentage of parasitized erythrocytes was determined from Giemsa-stained thin smears. Culture supernatant fluid, consisting of 40% normal bovine serum60% medium 199 supplemented with 15 mM 4-(2-hydroxyethyl-1-piperazine-ethanesulfonic acid [HEPES]), 100 ,ug of *

Corresponding author.

t Present address: Laboratory of Parasite Immunology, Department of Biology, University of Puerto Rico, Rio Piedras, PR 00931.

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VOL. 25, 1987

DETECTION OF B. BOVIS EXOANTIGEN

2.1 2 1.9 1.8 1.71.6 1.5 1.4 1.3 1.2-

concentration, 1 mg of total protein per ml) were assayed. The reaction was allowed to proceed for 2 h at 37°C in a humidified chamber. Subsequent steps were done as previously described. (ii) Reactivity with normal IgG. The assessment of the reactivity of normal bovine IgG with sample antigens was done by using nonimmune bovine IgG as capture antibody. (iii) Cross-reactivity with common pathogens. The crossreactivity with common pathogens of domestic animals was tested by substituting B. bovis exoantigens with the following: supernatant fluids and cell lysates from cultures of Anaplasma marginale and Babesia bigemina; homogenates from Dirofilaria immitis adult worms; and lysates of Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli cultures. Reactivity between homologous and heterologous B. bovis strains was examined by using anti-B. bovis IgG to the Venezuelan strain as a capture antibody and conjugate and by measuring the specific activity of the homologous compared with the heterologous culture-derived B. bovis exoantigens. To establish the kinetics of exoantigen release, supernatant fluids collected from B. bovis cultures at 24, 48, and 72 h were tested. After centrifugation, the initial protein concentration of all samples was adjusted to 1,000 ,ug/ml and five 10-fold dilutions were assayed for activity.

37 C 1 h

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-

*0.9 0.8 0.70.6 0.5 0.40.30.20.1 100

4 C 16h

-

0.1-

50

10

5

CAPRJRE *411800 CONCENTRAIO

2.5

1

(Rg/mI).

FIG. 1. Coating efficiency of anti-B. bovis IgG onto microtiter plates at 4 and 37°C. O.D., Optical density.

color development. For the two-site assay, 100 ,ul of capture antibody at an optimal concentration of 10 ,ug of total protein per ml in 0.05 M carbonate buffer, pH 9.6, was applied to the wells of flat-bottom polystyrene Immunolon 1 microtiter plates (Dynatech Laboratories, Inc., Alexandria, Va.) and the plates were incubated for 1 h at 37°C. Wells were washed five times with 0.2% Tween 20 in 0.15 M phosphate-buffered saline (PBS), pH 7.2, treated with 200 ,ul of 2% gelatin (Sigma) in PBS for 1 h at 37°C, and washed as described above. Then, 100 pi1 of test sample in PBS-0.05% Tween 20 (PBST) at an initial protein concentration of 2.5 mg/ml was added and incubated for 1 h at 37°C. After the wells were washed, 100 pi of horseradish peroxidase-conjugated specific anti-B. bovis IgG at an optimal dilution of 1:800 in PBST was added to the plates and allowed to react for 1 h at 37°C. After the plates were washed four times with PBST and then once with PBS only, 100 ,ul of 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) substrate was added. After 45 min, color development was determined at 414 nm with a Titertek multiscan MC microplate reader (Flow Laboratories, Inc., McLean, Va.). The results were expressed either in absorbance values or in EIA units on the basis of positive-negative ratios. Experiments involving optimization of the test made use of six replicates with two subsamples (n = 12). Experiments for antigen detection had three replicates with two subsamples (n = 6). The level of significance was established at 0.05%, and one-way analysis of variance was done by the method of Steele and Torrie (20). The optimal coating time and concentration of capture antibody were determined by a checkerboard titration. Specificity and sensitivity of the assay. The specificity of the assay for culture-derived B. bovis exoantigens was examined by the following steps: (i) inhibitory capacity of unlabeled specific IgG, (ii) reactivity with enzyme-conjugated normal IgG, and (iii) cross-reactivity with common pathogens of domestic animals. (i) Inhibition of the reaction. After reacting the capture antibody with culture-derived B. bovis exoantigens, five 10-fold dilutions of unlabeled bovine anti-B. bovis IgG (initial

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RESULTS AND DISCUSSION Plates incubated at 37°C for 1 h gave significantly higher readings at an optical density at 414 nm than did those incubated at 4°C for 16 h (Fig. 1). The coating efficiency indicated that bovine IgG in the two-site EIA behaved similarly to that described for human and rabbit immunoglobulins (7). Figure 2 demonstrates that 10 pg of IgG per ml gave the best results of any capture antibody concentration. At this concentration, the reaction produced an optimal positivenegative ratio. Therefore, an incubation period of 1 h at 37°C with 10 ,ug of capture antibody per ml was routinely used. 6

5

4

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25

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2.5

CATURE ANT1OODY CONCENTMTION

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1

0.1

Wg/mI)

FIG. 2. Effect of various capture antibody levels on EIA activity with a fixed concentration of culture-derived B. bovis exoantigen.

J. CLIN. MICROBIOL.

MONTEALEGRE ET AL.

1650 2.1

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1:20

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1:160

1:320

1:640

100

1:1280

50

ANTIGEN DILUTION

25

10

SAMPLE CONCENTRAlON

5

2.5

(Mg/mi)

FIG. 3. Assay of cross-reactivity of B. bovis and B. bigemina exoantigens with anti-B. bovis IgG. O.D., Optical density.

FIG. 5. Comparison of EIA reactivity of immune and normal IgG with culture-derived B. bovis exoantigens. O.D., Optical density.

In establishing the specificity of the test, a number of antigens, e.g., A. marginale, D. immitis, S. aureus, Streptococcus pyogenes, E. coli and B. bigemina, were tested. Only the antigens derived from B. bigemina cross-reacted significantly with anti-B. bovis bovine IgG (Fig. 3). Serological cross-reactivity has been previously documented with the indirect fluorescent-antibody (18), complement fixation (12), indirect hemagglutination (4), and latex agglutination (13) tests. Apparently, one or more antigens may be shared by B. bovis and B. bigemina (11). The specificity of the test was further assessed by an inhibition assay by using unlabeled anti-B. bovis IgG. A dose-dependent inhibition of the reaction was demonstrated (Fig. 4). The highest degree of inhibition was achieved at 0.1 to 1.0 Ftg of unlabeled specific IgG per ml. Normal bovine IgG gave negligible reactivity with B. bovis exoantigens (Fig. 5). The titration of culture-derived exoantigens obtained from different B. bovis isolates is depicted in Fig. 6. Significant activity was found in all the strains tested, with the homologous system giving the highest optical density readings.

This is consistent with the previous findings of common antigens among strains from various geographic regions (14). The degree of EIA reactivity of culture-derived exoantigens released in relation to a 72-h parasite cycle is depicted in Fig. 7. Samples collected at 24 h (percentage of parasitized erythrocytes = 6%) gave higher EIA values compared with those obtained at 48 h (percentage of parasitized erythrocytes = 9%) and 72 h (percentage of parasitized erythrocytes = 11%). This difference can be attributed to the rapid growth of the parasite in the first 24 h of in vitro culture as more exoantigens are released into the supernatant fluids.

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ANTIBODY CONCENTRATION (mg/ml)

FIG. 4. Inhibition of the EIA activity of culture-derived B. bovis exoantigens by unlabeled specific lgG antibody.

1:160

1:320

1:640

DILUTiON

FIG. 6. Titration of culture-derived exoantigens obtained from B. bovis strains isolated from Venezuela, Argentina, and Ecuador. Asterisk by Venezuela indicates capture antibody and conjugate prepared from the IgG fraction of hyperimmune anti-B. bovis (Venezuela strain) serum. O.D., Optical density.

VOL. 25, 1987

DETECTION OF B. BOVIS EXOANTIGEN

TABLE 1. Analysis of EIA test reproducibility: detection of antigens in B, bovis culture supernatant fluids collected and stored under different conditions

7

Condition of supernatant fluid testeda

Pooled Lyophilized Freshly collected Cultured 24 h 48 h 72 h

4 E

3

hi

2

a

48h

0

50

100

25

10

SMPLE CONCDTON

5

1

(