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1. Virus growth in tissue culture
2. Virus harvest
3. Inactivation
4. Concentration
5. Purification
6. Innocuity testing
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PROCEDURE FOR PRODUCING INACTIVATED, PURIFIED FMDV ANTIGEN
1. Virus growth in tissue culture
Baby Hamster Kidney (BHK) cells are commonly used for production of bulk
amounts of FMDV. Monolayer cultures are prepared, the supernatant fluid
is discarded and the cells are washed with PBS, which in turn is discarded.
The BHK cells are infected with an inoculum of FMDV containing 7-8 logs
TCID50/ml adsorbed at 37°C for 30 min, after which a minimum quantity
of serum-free maintenance medium is added (e.g., 20 ml Eagle's/10% Tryptose
Phosphate Broth per Roux bottle). Alternatively, virus can be added
to the maintenance medium on the monolayers, without an adsorption step.
Cultures are then incubated (rocked or rolled) at 37°C.
A similar protocol using a pig kidney cell line, IB-RS-2, is used for
the production of SVDV antigen.
2. Virus harvest
Cytopathic Effect (CPE) (Cytopathic Effect in Tissue Culture) should be
complete within 24 h. The supernatant fluid is harvested and clarified
at approximately 10 000g for 30 min, in a suitable high speed centrifuge.
An aliquot can be titrated to assess that the titre is = or preferably
>7 logs TCID50/ml.
3. Inactivation
Antigen is inactivated with 0.001 M binary ethyleneimine (BEI) at 37°C
for 24 h.
CARE! - AZIRIDINES (BEI) ARE CARCINOGENIC
Use BEA (2-bromoethylamine hydrobromide) mol wt 204.9.
a. Prepare 0.1 M BEI e.g., 0.205 g BEA by dissolving in 10 ml 0.2 N sodium
hydroxide.
b Incubate at 37°C for 1 hour to produce BEI.
c. Use 10 ml of 0.1 M BEI for 1000 ml of virus harvest (i.e., 0.001M BEI
final concentration) and leave at 37°C for 24 hours to inactivate antigen.
Ensure that all supernatant fluid comes into contact with BEI. Neutralise
any remaining BEI solution and tips, etc., which have been in contact
with BEI with 1% sulphuric acid solution.
4. Concentration
Concentrate the antigen using either (a.) 7.5% polyethylene glycol (PEG),
stirring at 4°C for at least 4 hours or (b.) precipitation with saturated
ammonium sulphate solution.
In either case deposit precipitates by centrifugation at approximately
10000g for 30 min and resuspend in a small volume of Tris buffer (0.05
M Tris, 0.1 M NaCl, 0.1% sodium azide, pH 7.6).
5. Purification
a. Clarify to remove insoluble material at approximately 3500g in a bench
centrifuge.
b. Pellet the virus antigen at 80 000-11 0000g for 2-4 hours.
c. Add 1-2 ml of Tris buffer to the pellet, and sonicate or agitate to
resuspend.
d. Remove insoluble material by centrifugation at 3500g for 20 min.
e. Prepare 15-45% sucrose gradients. Add a 1/10 dilution of 10% Nonidet
P-40 to the virus suspension (i.e., a final concentration of 1% Nonidet
P-40) and apply virus material to gradients. Centrifuge at 11 5000g for
4 hours using an appropriate swing-out rotor in a suitable ultracentrifuge.
f. Collect the 146S peak and quantify with a spectrophotometer (read at
259 nm; 1 OD unit = 132 µg/ml using 1 cm2 cuvette). Dispense in glass
containers treated with a water repellent (eg 'Repelcote') and store at
-70°C or above liquid nitrogen.
Antigen produced is appropriate for serum production and antigen detection
ELISA.
Varying degrees of success have been achieved using this procedure for
SVDV.
Alternatively the PEG stage (see 4 above) can be omitted after inactivation
and instead virus can be pelleted by ultracentrifugation, resuspended
in Tris buffer, and added to sucrose gradients (see 5b-f).
6. Innocuity testing
Virus inactivation can be verified by the method of Anderson et al., 1970
using BTY cells instead of BHK cells (Ferris
and Donaldson, 1984). A 5% volume of the inactivated virus preparation
is assessed for the presence of infectious virus by the inoculation of
aliquots onto monolayer cultures of BTY cells grown in either tubes or
flasks (75 cm2).
a. Wash cells with PBS immediately before inoculation with 0.2 ml of sample
per tube or 1 ml per flask. Leave cultures stationary at 37°C for 1 hour
to enable any live antigen present to adsorb to the cell sheet.
b. Add either 2 ml or 10 ml of Eagle's maintenance medium without serum.
c. Roll tubes or rock flasks at 37°C and observe cells microscopically
for evidence of CPE for 72 hours.
d. Harvest the supernatant fluid from first passage cultures after freeze-thawing
cultures, clarify and pool.
e. Inoculate aliquots onto fresh cultures (second passage); 1 ml per tube,
5 ml per flask i.e., half the volume of the first passage harvest pool.
Leave cultures stationary at 37°C for 1 hour and then wash monolayers
several times with PBS to eliminate the inoculum. Add maintenance medium
and return to 37°C as before.
f. Observe cultures microscopically for evidence of CPE for a further
72 hours.
g. If, after second tissue culture passage, there is no evidence of CPE,
the antigen preparations are deemed to be innocuous.
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