Immunisation protocol:
Immunisation was carried out according to
the following timescale:
-
Day 1: Preimmune bleed.
ELISA is carried out on preimmune serum. First immunisation with antigen in FCA
(Freund’s Complete Adjuvant), 10 intracutaneous injections into zones of
popliteal and inguinal lymph nodes.
-
Day 28: First boost
immunisation with antigen in FIA. 10 intracutaneous injections into zones of
popliteal and inguinal lymph nodes.
-
Day 42: Second boost
immunisation with antigen in FIA. 10 intracutaneous injections into zones of
popliteal and inguinal lymph nodes.
-
Day 52: First bleed; 50ml of
blood collected from 1 rabbit. Third boost immunisation with antigen in FIA. 10
intracutaneous injections into zones of popliteal and inguinal lymph nodes.
-
Day 62: Second bleed; 50ml
of blood collected from 1 rabbit.
-
Titer assay with ELISA on
immune serum. Alternative protocols can also be used according specific
requirements.
-
Customer receives preimmune
serum and IgG suspension in 33% ammonium sulphate.
Antibody isolation protocol:
Serum from each animal was treated
separately.
Serum was separated by centrifugation.
Serum was gathered, transferred to beakers, and saturated solution of ammonium
sulphate (pH 7.0) was added slowly up to 33% of saturation followed by
continuous mixing during 6h at +4º C. The suspension was centrifuged and the
pellet was washed twice with 33% solution of ammonium sulphate. Final pellet was
resuspended in 2-4ml of 33% solution of ammonium sulphate.
Storage: Ammonium sulphate
suspension: + 4º C, indefinitely long. Do not freeze! IgG solution substituted
with PBS and 50% glycerol can be stored at –20 - -70 °C for years. Preimmune
serum can be stored in small aliquots at -70 °C or for short period (weeks) with
antibacterial agents (e.g. sodium azide or thimerosal, 0.02%) at +4 °C.
IgG recovery: Adjust concentration
of ammonium sulphate up to 55% saturation by adding 0.11g of ammonium sulphate
powder or 0.21 ml of saturated solution (pH 7.0) to each 1 ml of IgG suspension.
Mix gently to dissolve the salt, incubate at +4
°C
for 1h. Pellet protein crystals by centrifuging and dissolve in an appropriate
buffer, e.g. PBS containing 50% glycerol for long-term storage.
Titer: A minimum titer was
detemined by direct ELISA. Titer was defined as the dilution of antibody
solution sufficient to give a change in absorbance of 1.0 at 450 nm after 20
minutes of substrate conversion at 25 °C. A microtiter plate was coated with
antigen at a concentration of 5 µg/ml in 0.05 M carbonate-bicarbonate buffer, pH
9.6. Colour reagent: 3,3′,5,5′-Tetramethylbenzidine (TMB).
Volume of antiserum taken:
2x25ml
(2x50ml of blood) from each rabbit. Rabbits will be kept for further 2 weeks should additional
buster immunisation and bleeding is required.
