Serological Survey of Epizootic Fish Viral Diseases in Some Rearing Rainbow Trout Farms by Enzyme-Linked Immunoabsorbent Assay (Elisa) in Iran

Blood Samples Preparation

Fifty-three serums of rainbow trout broodstocks (29 female and 24 male) were taken for detection of antibodies against IHNV, IPNV and VHSV that were collected during March till November 2003 from 13 hatcheries and rearing farms in three provinces in Iran Blood samples were taken from caudal vein of suspected broodstocks and it was clotted at room temperature. The clotted blood was centrifuged at 2,000 to 3,000 rpm for approximately 15 minutes. Then separated serum was transferred to Eppendorf tubes. Also in order to confirm virology examination two samples that had revealed CPE were chosen. Nine samples were omitted for some technical problems such as hemolysis. Finally, 44 serum specimens were transferred in sturdy, leak-proof plastic vials and stored at -20ºC freezer.

The Procedure of ELISA Examination

In the current study, Sandwich ELISA examination was selected for detection of antibody against of IHN, VHS and IPN in elected rainbow trout farms.

ELISA examination for antibody detection against IHNV in broodstock serum consists of the preparation of 1/180 dilution for inactivated IHNV antigen for the coating operation. According to manufactory protocol, 100µl of viral antigen was used for coating. Therefore 70 wells consist of 44 suspected samples; negative control and blank well were selected from an ELISA 96 plate for the coating operation. Required viral agents were calculated according to formula as follows: Request IHNV antigen = 70 (sample) × 100 µl (antigen) = 7000µl Recommended dilution = 1/180 Total necessary inactivated viral antigen = 7000 ÷ 180 ≈ 40 µl In fact, 40 µl inactivated viral antigen were needed so for preparation of request solution with 1/180 dilution it should be solved in 7ml (7000 µl) of carbonate buffer [8].

Mohammad Jalil Zorriehzahra, et al. Serological Survey of Epizootic Fish Viral Diseases in Some Rearing Rainbow Trout Farms by Enzyme-Linked Immunoabsorbent Assay (ELISA) in Iran. Cell Cellular Lif Sci J 2019, 4(1): 000139.

Well Selection for the Viral Antigen Coating Operation

In order to examine primary stages and initial set up, four binary rows were selected. So eight rows were chosen for viral antigen coating and altogether 64 wells were coated (16 × 4 = 64). Then 100 µl of diluted viral antigens was added to each well of ELISA plate with 1/180 dilution. Consequently, sample plates were covered with parafilm to prevent evaporation and then refrigerated (4ºC) for one day in favour of completion of coating procedure. After that ELISA plate was removed from the refrigerator on next day and discharged well contents completely. Then wells were washed with 250µl carbonate buffer (Coating buffer) for three times and discharged wells contents completely (Dilution of buffer was 0.25%). Then 250 µl of Bovine Serum Albumin (BSA) 3% were added to each well. Again sample plates were covered with parafilm and then stored at room temperature (22ºC) for one hour. Also, wells were washed with 250µl PBS Tween solution four times and discharged wells contents completely. In this stage wells, content discharge was done after 1 -2 minutes. {PBS tween = 1000 ml PBS+ 0.5 ml tween20}

Dilution Preparation of Suspected Serum Samples

The dilution of the whole 44 suspected serum samples were 1:2 and it was made as follows:

Test sample = (100μl serum sample + 100μl PBS Tween). Then 100 µl of diluted serum samples with 1:2 dilution was added to each well of ELISA plate and then were covered with parafilm and incubated at 25ºC for 1.5 hours. Eight serums were collected from good health condition farms in the same provinces and selected it as negative control sera. These serums were examined in virological examination and IFAT versus IHNV and no CPE was observed and findings were negative.

Then ELISA plate was removed from the incubator and after discharge of its contents, it was washed with PBS Tween four times. Consequently, Anti Rainbow trout IgM MAbs removed from -70ºC freezer and kept in room temperature. Next 100 µl was added to each well after MAbs melting. Then it was covered with parafilm and incubated at 25ºC for 1.5 hours. Then the plate was removed from the incubator and it was washed with PBS Tween. Next 100 µl of Anti-mouse IgG HRP conjugate which was diluted 1/3000 were added to each well and stored at room temperature at least for one hour. After Copyright© Mohammad Jalil Zorriehzahra, et al.

that 100 µl substrate solutions were added to each well. It was used pure and no need to dilute. After ten minutes the special reaction was occurred and some color changes in positive cases were observed. These changes were in the range of light blue till dark blue and mentioned color changes were observed in some wells and were recorded (Figure 1).

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Consequently, 50 µl stop solution was added after ten minutes to each well. This solution was changed blue color to yellow color in positive cases. The rate of change of color was the same in the last stage and depended to blue color in each well. In negative cases that blue color was not observed so yellow color was absent automatically (Figure 2).

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In the final stage, the ELISA plate arrived in ELISA reader machine and all Optical Density (O.D) was Mohammad Jalil Zorriehzahra, et al. Serological Survey of Epizootic Fish Viral Diseases in Some Rearing Rainbow Trout Farms by Enzyme-Linked Immunoabsorbent Assay (ELISA) in Iran. Cell Cellular Lif Sci J 2019, 4(1): 000139.

obtained. Then the result could be printed. For easy analysis, it was recommended that change in colors should be marked by a circle around of good number.

The first stage of ELISA optimization for antibody detection against IPNV and VHSV in brood stock serum consists of Protein measurement of IPNV and VHSV antigens. First of all, protein measurement of IPNV and VHSV antigens should be estimated that it as measured by Bio Photometer (Eppendorf®, 739 models) and its procedure carried out as follows:

Carbonate buffer (pH=9.6) was used as a BLANK in the test. For the preparation of the first dilution (1/60) 10 µl inactivated IPNV was solved in 590 µl Carbonate buffer. Therefore:

(10+590=600), 10/600 = 1/60 dilution.

The rate of requested viral antigen protein was 0.020 and its protein was measured by Photometer and it was 0.067 (about three times of requested rate = 0.020) so it was diluted three times (So the ratio of 0.060 reached to 0.020). Then 5 µl of viral antigen was solved in 895 µl carbonate buffer and 1/180 dilution was obtained. The same method was performed for VHSV and 1/180 dilution produced for both of them.

Sixteen serums were collected from good health condition farms in the same provinces and selected as negative control sera. Eight sera for IPNV and eight sera for VHS were specified. These serums were examined in virological examination and IFAT versus IPNV and VHSV and no CPE was observed and findings were negative.

For coating operation, 100 µl of viral antigens diluted (IPNV & IHNV) (with 1/180 dilution) were added to each well (Rows: A-H). Then a plate of ELISA was covered by parafilm and stored at 4ºC refrigerator for one day in favor of high completion of coating procedure. Buffers washing (with high and low salt) were carried out and all wells washed according to last protocol. The ELISA plate was removed from the refrigerator on the next day and discharged well contents completely. Again wells were washed with 250 µl low salt solution for three times and discharged wells contents completely. Then 50 µl of Bovine Serum Albumin (BSA) 3% were added to each well. The sample plates were covered with parafilm and then stored at room temperature (22ºC) for one hour. Again wells were washed with 250 µl low salt solution four times and discharged wells contents completely.

Copyright© Mohammad Jalil Zorriehzahra, et al.

Sample Selection Stage

Suspected serum samples were diluted with 1:1 dilution with PBS Tween. (100 µl serum sample + 100 µl PBS Tween). Serum samples from 1-7 for IPNV and 38-44 for VHSV were selected and 100 µl suspected serum was added to each well of ELISA plate and then were covered with parafilm and incubated at 25ºC for 1.5 hours.

The ELISA plate was removed from the incubator and after discharge of its contents; it was washed with high salt solution five times. Next 100 µl Anti Rainbow trout IgM MAbs were added to A, B, C, D, E, F, and H wells. G well-kept empty for positive control.

Positive Control Stage

A row of G well considered as a positive control in premature procedure with using of a polyclonal antibody that had produced before through inactivated viral antigens injection to rabbit but it was not ready so using of the positive control was decided for all samples.

Negative Control Stage

The negative control group is essential for comparison and analysis of findings in ELISA examination. Therefore 24 sera samples were collected from five same farms in mentioned provinces with high-quality health status. All fish have been satisfied health and no clinical signs were observed in the selected population.

All sera were examined in virology and IFAT assay and results were divided into three categories separately for three mentioned viral diseases. Then collected sera distributed in small vial aliquots and kept in -20ºC freezer. After that ELISA plate was covered with para film and stored at room temperature for one hour. All wells were washed with a high salt solution for five times. Anti- mouse IgG HRP conjugate (100 µl) with 1/3000 dilution was added to each well and incubated at 25ºC for 45-60 minutes. Again all wells were washed with high salt solution same last stage. Then 100 µl substrate solutions were added to each well. Primary results revealed that blue color changes were not observed for IPNV after 10 minutes. In wells of 40 and 42 reaction of blue color changes were observed for VHSV after 1-2 minutes. Then 100 µl stop solutions were added to each well and yellow color again was observed in wells of 40 and 42. In the final stage, ELISA plate arrived in ELISA reader machine with 450 wavelength nm and all O.D was obtained and results were printed.

Mohammad Jalil Zorriehzahra, et al. Serological Survey of Epizootic Fish Viral Diseases in Some Rearing Rainbow Trout Farms by Enzyme-Linked Immunoabsorbent Assay (ELISA) in Iran. Cell Cellular Lif Sci J 2019, 4(1): 000139.

Main ELISA Examination for Antibody Detection against IPNV and VHSV in Broodstock Serum Coating Stage for Viral Diluted Antigens

In purpose of good coating, 30 µl of IPN viral antigen were solved in 5.5 ml carbonate buffer and obtained 1/180 dilution for coating. Similar to the above stage, again 30 µl of VHS viral antigen were solved in 5.5 ml carbonate buffer and obtained 1/180 dilution for coating. Then 100 µl viral diluted antigens (IPNV and VHSV) with 1/180 dilution were added to each well and stored at 4ºC for coating operation overnight. After that suspected serum samples were distributed in ELISA plate but some serum samples such as numbers 5, 6, 13, 14, 15, 18, 19, 26 and 26 were removed for several reasons (serum hemolysis and serum low amount). Samples No = 40 and No= 42 were divided to more dilutions ¼, ⅛, 1/16, 1/32, 1/64 and 1/128 (because they had shown before some blue color changes in the optimization stage).

The ELISA plate was covered by parafilm and stored at 4ºC for overnight in favor of high completion of coating procedure. The ELISA plate was removed from the refrigerator on the next day and discharged well contents completely. After that wells were washed with 250 µl low salt solution for three times and discharged wells contents completely. Then 250 µl of Bovine Serum Albumin (BSA) 3% were added to each well and stored at temperature room for one hour. Again wells were washed with 250 µl low salt solution four times and wells contents discharged completely.

Sample Adding Stage

First of all 100µl suspected serum samples were added to each well which had been diluted with 50/50 dilution with PBS tween (100 µl serum sample + 100 µl PBS tween). Then ELISA plate was covered with parafilm and incubated at 25ºC for 1.5 hours. Next, ELISA plate was removed from incubator and wells were washed with 250 µl high salt solution five times and discharged wells contents completely.

Anti-Rainbow Trout IgM MAbs Adding Stage

First of all, 100 µl Anti Rainbow trout IgM MAbs were added to all wells and also blank well. Then 100 µl PAbs were added to special well (Rabbit Hyper Immune Serum) it had produced before through injection to the rabbit. (This antibody were diluted 1/20 with PBS tween). Then ELISA plate was covered with parafilm and stored at room temperature for one hour. Next, all wells were Copyright© Mohammad Jalil Zorriehzahra, et al.

washed with 250 µl high salt solution five times and discharged wells contents completely (last time was extended 5 minutes). After that 100 µl of Anti-Mouse IgG HRP conjugate with 1/2000 dilution were added to each well (It had before diluted in PBS tween). Consequently, 100 µl of Anti-Rabbit IgG HRP conjugate with 1/20000 dilution was added to a well of Rabbit Hyper Immune Serum (this conjugate had been diluted in PBS tween). Then ELISA plates stored at room temperature for 1 hour. Next, all wells were washed with 250 µl high salt solution five times and discharged wells contents completely (last time was 5 minutes). Next 100 µl substrate solution were added to all wells and waited for 15 minutes. Some blue color changes reaction were observed in some wells that it considered as positive samples. Then 50 µl stop solution were added to all wells and yellow color changes were observed in blue wells. In the final stage, ELISA plates arrived in ELISA reader machine with 450 nm wavelengths and all O.D findings were printed.

When 100 µl substrate solutions were added to wells of Rabbit Hyper Immune Serum after a few seconds a blue color reaction was observed immediately that it could be revealed this ELISA examination was desirable and optimization was done in good condition.