Indirect Enzyme-linked Immunosorbent Assay (ELISA) for the
Detection of Eel Serum Antibody
Takaji Iida, Hiroyuki Yonekura, Masafumi Izumiyama
and Hisatsugu Wakabayashi
Department of Fisheries, Faculty of Agriculture, The Uniersity of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan
(Received July 20, 1991)
Immunoglobulin (Ig) was purified from the serum of eel (Anguilla japonica) and an indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of eel antibody. Sera were collected from eel which had been injected with formalin-killed Edwardsiella tarda bacterin. A pool of the sera was precipitatedwith a 40% saturated ammonium sulfate solution , and then immunoglobulin was purified from the precipitates by gel-filtration and ion-exchange chromatography . The purified Ig emulsified in Freund's complete adjuvant was administered to a rabbit to prepare anti-eel Ig serum .
Eel were immersed in the E. tarda bacterin at a density of 1 mg wet weight/ml for 1 h . Change in antibody titers in the vaccinated fish was determined by indirect ELISA . The titers of vaccinated fish were significantly higher than those of unvaccinated control fish .
It is generally recognized that antibody responses are so weak in fish vaccinated by oral or immersion administration that the antibodies are not always detected by aggluti nation technique. Recently, the enzyme-linked immunosorbent assay (ELISA) , has been widely used for detection of antibodies as well as antigens. Smith (1981) used ELISA for detec tion of Aeromonas salmonicida in diseased fish tissue. Roberson (1981) detected the fish antibody by thin-layer ELISA. Thuvander et al. (1987) demonstrated antibody production in rainbow trout vaccinated against vibriosis by immersion method. In the present study, we purified immunobulin (Ig) from Japanese eel (Anguilla japonica) serum and developed an indirect ELISA to detect antibody in the serum of the eel vacccinated by immersion against Edwardsiella tarda.
Materials and Methods
Eel Antiserum Preparation
Japanese eel weighing about 200 g were intraperitoneally injected with formalin-killed E. tarda SU226 bacterin at a dose of 1 mg wet weight/fish at an interval of one week.
Two weeks after the second vaccination, blood was taken from bulbus arteriosus and allowed to clot for 2 h at room temperature followed by storing at 4•Ž overnight. Serum separated by centrifugation was pooled from 91 fish and inactivated by heating at 50•Ž for 30 min. The agglutination titer of the pooled sera was 1:512.
Purification of Eel Ig
Precipitate was extracted from the eel anti serum with 20% saturated ammonium sulfate solution on ice and removed by centrifugation at 15,000g for 10 min at 5•Ž. The supernatant was re-precipitated with a final saturation of 40% ammonium sulfate solution on ice. The precipitate was resuspended in 0.01 M Tris-HCl buffer (pH 7.2), desalted by dialyzer (Zeineh Dialyzers model D-1, Funakoshi, Japan) and concentrated by ultrafiltration (Mini-Module model NM-3, Asahikasei, Japan) . Purification of Ig was performed with gel-filtration. A 2.6•~100 cm, Sephacryl S-300 Superfine (Pharma cia, Sweden) column was used at a flow rate of 20.0 ml/h with 0.01 M Tri-HCl buffer (pH 7.2) containing 0.15 M NaCl. The samples were collected at a volume of 4.4 ml/tube.
and a series of 2.6 ml fractions was collected.
Rabbitt anti-Eel Ig
A rabbit was subcutaneously injected with I mg protein of eel Ig emulsified in Freund's complete adjuvant (Wako Chemicals, Japan). Additional injection with the Ig at the same dose was carried out twice at intervals of one week. In additional injections, incomplete adjuvant was used instead of complete one. Antibody was checked by Ouchterlony test.
Antigen for Indirect ELISA
E. tarda SU226 was incubated on Trypto-Soya Agar (Nissui, Japan) at 25•Ž for 48 h, and suspended in saline at a density of 10 mg wet weight/ml. Bacterial cells were then sonicated and autoclaved at 121•Ž for 15 min. After centrifugation at 600g for 15 min, the supernantant was used as an antigen at 1:980 in 0.05 M carbonate buffer (pH 9.6) for indirect ELISA.
Indirect ELISA Procedure
Indirect ELISA was carried out by the modified method of Cossarine-Dunier (1985). E. tarda antigen (200 ƒÊl) was loaded on ELISA microplates (Flow Laboratories, USA), and incubated overnight at 4•Ž. Unbound antigen was removed by three successive washings with phosphate buffered saline (pH 7.2) con-taining 0.05% Tween 20 (PBS-Tween). After blocking for antigen-uncoated sites with 200 pi of skimmed milk solution (108 g/l) at 37•Ž for 1 h, 100 ƒÊl of diluted eel serum samples (1:81 with PBS-Tween) was added to appropriate wells, incubated at 30•Ž for 1 h, and washed three times with PBS-Tween. One hundred microliters of rabbit anti-eel Ig was added to the wells, incubated at 37•Ž for 1 h and washed. After that, 100 ƒÊl of goat anti-rabbit IgG peroxidase conjugate (Cappel, USA) diluted
0.1 M citric acid and 0.2 M sodium phosphate dibasic at a ratio of 1:1, pH 4.8. The reaction was stopped by adding 1.25% sodium fluorine (50 id). The resulting colour reaction was measured at 405 nm by using of a microplate reader (MPR A4, Tosoh, Japan).
Immersion Vaccination
The experimental eel had been raised from the glass eel stage in Fisheries Laboratory, Faculty of Agriculture, University of Tokyo, and no disease was recorded prior to the ex-periment. The fish were divided into four grougs: Groups, 1, 2 and 3 were immersed in E. tarda bacterin at a density of 1 mg wet weight/ml for 1 h once, twice and three times, respectively. The fourth group was immersed in saline for 1 h and kept as control. The vaccination was repeated at intervals of one week. Blood samdles for antibody analysis were taken one week after the last vaccination.
Results
Elution profile of gel-filtration and the ag-glutination titers are shown in Fig. 1. Four peaks of absorption at 280 nm were observed in gel-filtration and agglutinating activity was detected in the first peak. The fractions from
Fig. 1. Elution profile by gel-filtration and the agglutination titer.
34 to 43 in which the activity was detected were pooled and applied on ion-exchange chromatography.
Figure 2 shows the results of the first ion-exchange chromatography. Agglutination was detected at fractions from 40 to 70. Because of low concentration of protein and wide elution, the fractions from 41 to 63 were again pooled and applied on ion-exchange chromatography in the same manner.
By the second ion-exchange chromatography, two peaks were obtained. The first peak was at fraction no. 42 and the second one at no. 47 (Fig. 3). However, it was revealed by
polyacrilamide gel electrophoresis (PAGE) that both the first and second peaks consisted of the same Ig (Fig. 4). The fractions form 41 to 48 were pooled and used for rabbit anti-eel
Fig. 2. Elution profile by 1st ion-exchange chro-matography and the agglutination titer.
Fig. 3. Elution profile by 2nd ion-exchange chro-matography and the agglutination titer.
Fig. 4. PAGE of purified eel Ig by 2nd ion-ex-change chromatography.
Lane I, First peak; Lane 2, Between first and second peak; Lane 3, Second peak.
Fig. 5. ELISA titers by immersion vaccination. Cont., Unvaccinated control; G. 1, G. 2 and G. 3, Group of vaccination once, twice and three times, respectivily. Each points represents a single fish.
Fig. 6. Double diffusion precipitation against rab-bit and-eel Ig.
Center well, Rabbit anti-eel Ig; Well A. Eel whole serum; Well B, Purified eel Ig after 1st ion-exchange chromatography; Well C, D and E, Purified eel Ig after 2nd
ion-exchange chromatography (C, First
peak ; D, Between first and second peak ; E, Second peak).
respectively) . The mean titer of group 3 was significantly higher than that of group 1 (P< 0.05) .
There was no difference in agglutination t iter by microtiter method between all vaccinated groups and control group.
Discussion
Immunoglobulin is usually purified by com binations of precipitation with ammonium sulphate, gel filtration and ion-exchange chro matography. McArthur and Sengupta (1982) reported the purification of New Zealand short finned eel (Anguilla australis schmidtii) Ig, but they did not mention the method in detail. Fukuda and Kusuda (1982) purified yellowtail Ig by a gel filtration and two ion-exchange
chromatographies. They recommended the
use of precipitation at the first step. We tried to purify eel Ig according to their recommenda tion, and obtained good results in quality and quantity of purified eel Ig to prepare an anti eel Ig rabbit serum. At the final step of eel Ig purification, two peaks of absorbance at 280 nm were observed. But their molecular weight estimated by PAGE were found to be same and the double immunodiffusion precipi tation lines were coalescent (Figs. 4 and 6). The reason why two peaks were observed is not clear.
Currently, ELISA is one of the most popular techniques for the detection of antibody from animals infected or vaccinated with micro organisms. ELISA to detect fish antibodies was first reported by Roberson (1981). Sub sequently, Thuvander et al. (1987) measured the antibody titers by using ELISA after vac cination of the rainbow trout against vibriosis. Waterstrat et al. (1989) used an indirect ELISA to detect channel catfish antibodies to Edward siella ictaluri. In the present study, it was
ELISA it is important to reduce the background level of non-specific interactions. We used a sonicated and heat-inactivated bacterial extracts as an antigen for ELISA. This antigen prepa ration was thought to contain LPS (Cossarini
-Dunier, 1985). However, further improvement of the antigen preparation for ELISA is required and the border between positive and negative
ELISA titers should be established.
Acknowledgments
We express our appreciation to Dr. Y. Suzuki,
Department of Fisheries, Faculty of Agricul
ture, The University of Tokyo for useful
advices, and Dr. J. S. Rohovec, Department
of Microbiology, Oregon State University for
reviewing the manuscript. We also thank the
staffs of Fisheries Laboratory, The University of Tokyo for their kind help.
References
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Fukuda, Y. and R. Kusuda (1982) : Purification and characterization of yellowtail immunoglobulin. Nippon Suisan Gakkaishi, 48, 921-926. (in Japanese with English abstract.)
McArthur, C. P. and S. Sengupta (1982) : A rapid micro-method for screening eel sera for antibodies against the digenean Telogaster opithorchis Mac farlane, 1945. J. Fish Dis., 5, 67-70.
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