3.2.5.1. Antigen formulation
(a) GFP
Purified recombinant GFP (Millipore, Temecula, USA) from a solution of 300 µg in PBS (20
% glycerol) was diluted to stocks of 50 µg 100 µL-1 (0.5 mg mL-1) in PBS. After mixing the protein 30:70 in MontanideTM ISA 760 VG the final dose was 0.15 mg mL-1. The solution was emulsified by vortexing for 4-5 min prior to inoculating carp with a dose of 15 µg fish-1.
(b) KHV
An inactivated KHV vaccine was kindly provided by Dr. Ian Pardoe (Henderson Morley PLc., Birmingham, England - Jan 2009), which was developed through formalin inactivation of purified virus. The total dose of vaccine was 1.6 mg mL-1 purified KHV in aluminium hydroxide adjuvant.
3.2.5.2 Experimental design
An immunisation trial was undertaken to assess the feasibility of utilising GFP as an exogenous marker antigen in Mirror carp (Cyprinus carpio L.), which would not interfere with antibody responses induced by the KHV component in the vaccine. Mirror carp (n = 50) weighing between 30 – 40 g were obtained from a carp farm in Hampshire, England
(Hampshire Carp Hatcheries, Hampshire, England) with no history of KHV. The carp were maintained in fresh water at the Aquatic Research Facility (ARF) at the Institute of Aquaculture, University of Stirling, Stirling, Scotland in 100 L tanks of fresh dechlorinated (Elga Piltramat AC4) mains water on a flow through system at 14°C ± 2 with a flow rate of 1 L min-1 oxygenated through air stones (Betta, J and K Aquatics Ltd., UK). The fish were acclimated to these conditions for at least 4 weeks prior to commencing any experimental work. Five fish were pre-bled to screen for anti-KHV antibodies by antibody ELISA. For the vaccination experiment, 36 of the remaining fish were randomly divided into three groups of 8 and one group of 10 and vaccinated as described in Section 3.2.4.2 (a). One group of 8 carp were injected ip with 0.1 mLfish-1 ofadjuvanted recombinant GFP. The second group of 8 fish were injected ip with 0.1 mL of inactivated KHV vaccine. A third group of 8 fish received two ip injections of 0.1 mL adjuvanted GFP and 0.1 mL KHV vaccine, one injection either side of the ventral line. The final group of 10 fish were inoculated ip with 0.1 mL of adjuvanted PBS to serve as a negative control. Following vaccination the fish were transferred to a recovery tank of rearing water before being replaced into their respective holding tanks. The fish were fed ad lib on a commercial pellet diet (Skretting, Norway) and checked twice a day for any adverse reactions. After 6 wpv, fish were sampled for serological analysis as described for Atlantic salmon in section 3.2.4.2 (a).
3.2.5.3 GFP KHV marker vaccine ELISA
(a) ELISA optimisation
The GFP ELISA was optimised using a similar approach to that taken for the FITC and KLH ELISAs to screen salmon serum as described in Section 3.2.4.3.(a). The protocol used for screening carp sera for anti-GFP antibodies was considered optimised once the absorbance
for the anti-GFP MAbs had an absorbance OD450nm ≥ 1 and negative sera was below the sensitivity threshold.
Optimisation of the KHV ELISA was undertaken based on published protocols according to Adkison et al. (2005) and St-Hilaire et al. (2009) and the protocol developed by Dr. Sven Bergmann (pers. comm.) with modifications. Intensive blocking appeared critical as carp sera tended to produce a high level of non-specific binding, thought to be associated with natural antibodies. As positive and negative control sera was available, it was possible to perform checkerboard assays with sera using various blockers including casein, BSA and a synthetic blocker, Roti-block (Roth), until the minimal ODs from negative sera were obtained without affecting the endpoint titre of the positive control of 1/1600 (i.e. until the concentrations that generated the maximum positive to negative (P/N) ratio).
(b) GFP ELISA
Immulon-4 HBX 96-well microtitre plates were coated with 100 µL of 20 µg mL-1 of purified recombinant GFP (Millipore) well-1. The plate was incubated at 4°C overnight then washed 3 x with LSWB and post-coated to block non-specific binding. Blocking methods differed for GFP protein and KHV antigen as signals from carp sera on the GFP ELISA were always much lower compared to that of KHV coated plates during optimisation. The plate was blocked with 5% casein in H2O for 3 h at RT then washed 3 x with LSWB. One hundred microliters per well of 2-fold dilutions of carp sera from 1/100 to 1/1600 diluted in PBS was added to the plate in duplicate and incubated overnight at 4°C. Positive antigen control wells received 100 µL of 4 µg mL-1 in duplicate of anti-GFP MAbs (Roche, Mannheim, Germany) diluted in PBS. Negative control blank wells received 100 µL PBS instead of carp sera. The following day the plate was washed 5 x with HSWB and 100 µL well-1 anti-carp IgM mouse IgG MAbs (Aquatic Diagnostics, Stirling, UK) diluted 1/55 were added to the plate and
incubated at RT for 1 h. The plate was washed again 5 x HSWB with a 5 min incubation on the final wash and the remainder of the assay was undertaken identically to that used for the detection of anti-FITC, anti-KLH and anti-ISAV antibodies (Section 3.2.4.3).
(c) KHV ELISA
Immulon-4 HBX 96-well microtitre plates were coated with 50 µL of sucrose purified KHV (Section 2.5.2) or BSA at 0.3 µg well-1 in 0.05M carbonate-bicarbonate buffer (Sigma-Aldrich) and incubated overnight at 4oC, followed by three washes with LSWB. Non-specific binding sites on wells were blocked with 250 µL of 10 % casein (w/v) in distilled water for 5 h at RT, before being washed with LSWB and adding 50 µL of either two-fold serial dilutions of vaccinated Mirror carp serum samples from 1/200–1/3200 or positive and negative control serum diluted in 5 % casein in PBS, as well as 5 % casein in PBS to two duplicate wells on each plate as a blank negative control. Pre-bled Mirror carp prior to vaccination were used for negative sera controls and anti-sera from infected koi from an experimental challenge undertaken at the Centre for Environment, Fisheries and Aquaculture science (CEFAS) with an antibody titre of 1/1600 (kindly provided by Dr. Peter Dixon) was used as a positive control. After incubating the sera on the ELISA plate overnight at 4oC, wells were washed 5 x HSWB with 5 min incubation on the last wash. Fifty microlitres of mouse anti-carp IgM diluted 1:73.3 in 0.1% BSA in PBS was added to each well and incubated for 1 h. Wells were washed with HSWB and incubated with 50 µL of goat anti-mouse IgG-HRP Conjugate (Sigma-Aldrich) diluted 1:4000 in 0.1% BSA in LSWB.
Following the wash step with HSWB, 100 µL chromogen (42 mM TMB in 1 part acetic acid to 2 parts distilled water) diluted 1:100 in substrate buffer (0.1 M citric acid, 0.1 M sodium acetate, pH 5.4, 0.033% H2O2) was added to each well. The reaction was stopped after 12 min by the addition of 50 µL of 2M H2SO4 well-1 and plates read at 450 nm using the Synergy™ HT Multi-Mode Microplate Reader (BioTek Instruments, USA).