Abstract

The processes and kinetics of the immune response in fish can be delineated through assays for specific antibody secreting cells produced in response to antigen stimulation. The traditional Jerne Plaque assay for detection of antibody secreting cells has been modified for use in fish. However, utilization of the Jerne plaque assay is limited by the obligatory use of lysable sheep red blood cells (SRBC) as indicators of antibody secretion, the necessity of coupling antigens to SRBC, the limited number of antigens capable of being coupled to SRBC and the presence in many fish of natural serum hemolysins to SRBC. The recent development of an ELISA based plaque assay (Czerkinsky et al, 1983; Sedgwick and Holt, 1986) has removed many of the constraints associated with the Jerne plaque assay, afforded an equal or greater sensitivity and has reduced both the time and labor required to conduct the assay. Procedures for the use of this assay have been adapted for use in fish by our laboratory. Briefly, the assay involves incubating catfish lymphocyte populations in 24-well plates previously coated with the antigen of interest. Cells producing antibody to the antigen leave an immunological fingerprint of bound antibody which is detected through the use of an ELISA technique. The procedure has been standardized and the specificity of the reaction demonstrated. Cells taken from fish vaccinated with a specific antigen exhibit the greatest response on wells coated with the antigen and minimal responses to other antigens. A strong positive correlation has been demonstrated between the number of Elisaplaques and serum agglutination titer. Using the ELISA plaque assay, we have been able to demonstrate that antibody secreting lympocytes locate at a different density interface or behave differently than other lymphocyte populations when separated through discontinuous Percoll gradient centrifugation. The use of the Elisa plaque assay has also allowed us to examine the distribution of antibody secreting cells among hematopoietic organs of the channel catfish. Among the organs the head kidney appears to produce more antibody secreting cells per million lymphocytes than do spleen or peripheral blood lymphocytes.

Introduction

The processes and kinetics of antibody responses can be delineated through assays for specific antibody secreting cells produced in response to antigen stimulation. The procedure allows an earlier and more detailed analysis of immune response than possible with ser-um antibody titer. In vitro studies of antibody secreting cells have provided information surrounding the effect of environmental perturbation and stress on immune function and has been essential in the development effective vaccines (Anderson, Dixon and Lizzio, 1986). Although Miller and Clem (1984) have developed a modification of the Jerne plaque assay for use in catfish, utilization of this assay has been limited by the obligatory use of lysable sheep red blood cells (SRBC) as indicators of antibody secretion, the necessity of coupling antigens to SRBC, the presence of natural hemolysins in catfish serum to SRBC, and the intensive time and labor required to conduct the assay. In mammals the development of an ELISA based plaque assays (Elisa plaque) for antibody secreting cells (Czerkinsky et al, 1983; Sedgwick and Holt, 1986) has removed many of the constraints associated with the Jerrie plaque assay and has afforded equal or greater sensitivity with a reduction in both the time and labor required to conduct the assay. Adaptation of this procedure for use in fish could significantly improve the ability to assess humoral responses among fish populations. Toward that end, we have adapted the Elisa plaque assay for use in channel catfish.

Materials and Methods

Experimental Animals

Channel catfish (Ictalurus punctatus Rafinesque) weighing 150 gm - 250 gm were maintained in 60 liter stainless steel flow-through tanks supplied with well-water maintained at a temperature of 20° + 1° C. The catfish were fed once daily at 2% total body weight. The three groups were randomly assigned to one of three treatments. Individual fish were given coded identification marks through intradermal injection of alcian blue dye with a Pan-jet inoculator (Panjet, Wright and Sons, Dundee, Scotland). Fish subjected to experimental treatment were immunized with three 0. 1 ml intraperitoneal injections at 7 day intervals of either Edwardsiella ictaluri whole bacterin in Freund's complete adjuvant (FCA); Staphlococcus aureus in (FCA); or Chicken red blood cells (CRBC) in HBSS. The whole bacterins were produced from 18 hour cultures of the respective bacteria grown in brain-heart infusion broth (BHI, Difco, Detroit, MI) at 27°C. The bacteria were washed 3 times in 0.01M phosphate buffered saline, pH 7.2 (PBS) and heat killed at 65°C for 60 min. Bacteria were mixed in FCA to a final concentration of 1 x 1010 bacteria/ml. Glutaraldehyde fixed chicken red blood cells were washed 3 times in HBSS and resuspended it a concentration of 7.1 x 10⁶ cells/ml in HBSS. Samples of peripheral blood and serum were collected from the caudal vein prior to each immunization and 14 days following the final immunization.

Cell Suspensions

Peripheral blood was obtained from experimental fish anesthetized with tricaine methanesulfonate (MS222) bled from the caudal vein with heparinized, evacuated tubes (Vacutainer, Becton Dickinson, Rutherford, N. J.). Following anesthetization and bleeding, the head kidney, and spleen were removed from individual fish and placed in calcium-magnesium free HBSS (CMFHBSS). To obtain single cell suspensions from head kidney and spleen, the respective organs were sieved and washed through a stainless-steel mesh (Cellector) using CMF-HBSS. Respective cell suspensions from peripheral blood, head kidney and spleen were then washed in CMF-HBSS at 300 g and layered over a discontinuous density Percoll gradient according to the method of Waterstrat, et al --1987. Following centrifugation at 300 g for 30 min, cells sedimenting at the 1.060 -1.065 gm/ml, 1.065 -1.070 gm/ml and 1.070 - 1.080 gm/ml interface were collected and designated as C1, C2 and C3 cells respectively. The suspensions were then washed and resuspended 90% RPMI -1640 supplemented with 10% heat- inactivated human serum, 5% heat inactivated bovine calf serum and 1% L-glutamine. The cells were counted with a hemocytometer and examined for viability using trypan blue dye exclusion. Cell viability was greater than 95%. The cell suspensions were characterized through replicate cytocentrifuge preparations stained with Wright's stain, non-specific esterase (15) or Sudan Black B (16).

Agglutination Titers

Sera to be tested for agglutination titer were subjected to two-fold dilution from 1:10 to 1:2560 in 1.0% bovine serum albumin-0.05M Tris buffered saline (BSA-TBS) against heat-killed bacteria or CRBC suspended in (PBS) according to modification the microtiter procedure of Herbert (1978).

Elisaplaque Assay Procedure

Bacteria or chicken red blood cells were attached to 24 - well tissue culture plates through modifications of the standard ELISA procedure outlined by Waterstrat et al 1989. To accommodate the increased volume of the 24 - well plate dispensed volumes were increased to 400 µl. Four hundred microliters of the catfish cell suspension at a concentration of 2.5 x 10⁶ cells per ml were added to each well and incubated for 2 hr at 27°C and 5% CO₂. Following incubation the cells were removed and the 24 -well plates washed 3 times in 0.01M PBS- 0.05% Tween. Next, 400 µl of the monoclonal antibody 9E1, specific for catfish immunoglobulin, diluted 1:4 in 1% BSA -TBS - 0.05% Tween was added to each well and allowed to incubate for 2 hr at room temperature. Following incubation, 9E1 was removed and the 24 -well plate washed 3 times in 0.01M PBS- 0.05% Tween. Four hundred microliters of Goat anti-mouse alkaline phosphatase diluted 1:500 in 1% heat- inactivated normal goat serum - TBS -0.05% Tween was then added to each well and incubated for 2 hr at room temperature. The conjugate was removed and the plates washed 3 times in 0.01M PBS- 0.05% Tween. Finally, 200 µl of a substrate-agarose mixture was added into the wells and allowed to develop for 30 min at room temperature and then stopped through the addition of 100 µl 3M NaOH. The substrate- agarose mixture was comprised of 5-BCIP 1 mg/ml in AMP buffer (Sedgwick and Holt, 1986) containing 1 mM levamisole mixed 4:1 with 1.5 % agarose. A positive reaction was indicated by the development of a blue insoluble precipitate. Controls used in the assay consisted of antigen coated wells and back-coated wells lacking antigen to which either cell suspension, Monoclonal 9EI, conjugate or substrate was added. The Elisaplaque assays were evaluated blindly without reference to the plating matrix of cells versus antigen using a dissection scope at a magnification of 25 X. Elisa plaque counts were corrected to account for differences in the proportion of lymphocytes among cell suspensions by dividing the raw counts by the proportion of lymphocytes as determined from differential counts as outlined above.

To demonstrate the specificity of the Elisa plaque procedure cells obtained from fish immunized with either E. ictaluri, S. aureus or CRBC were added to wells plated with the respective antigen. For example, cells from fish immunized with E. ictaluri were added to wells coated with E. ictaluri, S. aureus or CRBC; a specific response would be indicated by the presence of Elisa plaques in wells coated with E. ictaluri and minimal responses in wells coated with S. aureus or CRBC.

Results

The specificity of the Elisa plaque assay is demonstrated in Figure 1. Statistically significant differences (p =0.015) were observed among the Elisa plaque responses of immunized fish to antigen coated wells. Fish immunized with E. ictaluri, S. aureus or CRBC demonstrated the greatest response on wells coated with the immunizing antigen. Figure 2 presents the relationship between agglutination titer and the number of Elisaplaques generated to the respective antigens. Agglutination titer appeared to increase as the Elisaplaque response increased.

Figure 1. Specificity of the Elisaplaque assay. Mean number of Elisaplaques generated plotted against antigen coating for fish vaccinated IP with either Edwardsiella ictaluri, (E. Ictaluri) Staphlococcus aureus (S. aureus) or Chicken red blood cells (CRBC). The numbers represent the mean number of Elisaplaques generated per 5.0 x 105 head kidney lymphocytes (n=6) obtained from the 1.060 - 1.65 interface of a discontinuous Percoll gradient.

In examining the responses of fish immunized with E. ictaluri, significant differences were observed in Elisaplaque response among lymphocytes subpopulations sedimenting at the 1.060 1.065 gm/ml, 1.065 -1.070 gm/ml and 1.070 - 1.080 gm/ml interfaces with the greatest response occurring in lymphocytes sedimenting in the C1: 1.060 - 1.065 gm/ml density interface (Figure 3). Significant differences were observed among the responses of lymphocytes from peripheral blood, head kidney and spleen, with head kidney lymphocytes exhibiting the greatest response (Figure 4).

Discussion

Initial work with the ELISA plaque assay utilizing rabbit anti- catfish immunoglobulin as the primary antibody in the detection system was frustratingly plagued with the presence of high background or non-specific reactions. The addition of 1 mM levamisol to the substrate solution to block endogenous alkaline phosphatase and the incorporation of 1% normal goat serum in the diluent buffer for the GAR-Alkaline Phosphatase conjugate greatly alleviated the problem of nonspecific reaction. Further refinement of the assay was achieved through the incorporation of the 9E1 monoclonal antibody to channel catfish immunoglobulin developed and kindly provided by Dr. N. Miller, University of Mississippi Medical Center, Jackson MS. The original attempt to demonstrate the specificity of the assay using Aeromonas hydrophila was confounded by the occurrence of high antibody titer to A.hydrolphila among unimmunized experimental fish. Using the antigens Stalphlococcus aureus and chicken red blood cells, antigens which catfish rarely encounter, the procedure has been standardized and the specificity of the reaction demonstrated (Figure 1). Evidence from the Elisaplaque assay indicates that antibody secreting cells locate at a different density interface (Cl) or behave differently than other lymphocyte populations (Figure.3) when subjected to discontinuous Percoll gradient centifugation. Since macrophages also layer in the Cl: 1.60-1.065 interface and are minimally distributed in other density interfaces, further studies are being conducted to determine if high Elisaplaque response present in the C 1 layer is merely due to differences in the sedimentation coefficient of antibody secreting cells or due to the presence of macrophages acting as accessory cells in the production of specific immunoglobin. Among the lymphoid examined the head kidney appears to produce more antibody secreting cells per million lymphocytes than do spleen or peripheral blood lymphocytes. Current work is directed to examining the kinetics of antibody secreting cell distribution following immunization.

References

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