Biomedical Engineering Reference
In-Depth Information
(6)
Measure the protein released in the supernatant by BCA protein assay at vari-
ous times. Add equal quantity of fresh buffer after each sampling to maintain
the sink conditions. Take the volume and protein quantity from the portions
that were removed at each time point into consideration during calculations.
The freeze dry coating with trehalose indicated that HA core adsorbed 2.9% of
the trehalose giving a slightly negative zeta potential which confirmed the coat-
ing of sugar layer.
111
Adsorption loading of OVA gave negative zeta potential
value of −11.34 ± 1.4 mV. The release of OVA every 10 min shown indicated that
about 40% of the absorbed OVA was released into the medium. After 30 min,
subsequent release of OVA was small and decreased over time giving a cumula-
tive OVA release in 50 min at about 90%.
111
7.4.2.4 In Vivo Immunological Response
The aquasomes prepared in the previous section can be used to harness immu-
nological response in female BALB/c mice (age 7-9 weeks; weight 15-20 g).
The mice have to be properly housed (23 ± 2 °C; RH: 60%) with 12 h light-
dark cycle controlled animal quarters in group of six (
n
= 6) and fed standard
rodent pellet and have free access to drinking water. Immunizations can fol-
low after one week of acclimatization with the environment. This process was
published and implemented by Pandey's group
111
involved no food intake 3 h
before immunization. Animal immunizations regimen are described below.
(1)
The animals are immunized by two intradermal injections with two weeks
interval, i.e. at day 0 (priming) and at day 14 (boosting) with one of the
following: (i) free OVA (10 µg per mouse) in 50 µl of PBS; (ii) blank aqua-
somes 100 µg in 50 µl of PBS; (iii) OVA aquasomes (equivalent of 10 µg of
OVA per mouse); and (iv) OVA in alum (OVA alum) and (v) sterile PBS
50 µl only. OVA alum are prepared by dissolving the protein (50 µg/mL) in
PBS (pH 7.4, 0.01 M) and subsequently mixing by sonication with alumi-
num hydroxide gel (1 mg/mL).
(2)
Blood from the saphenous vein of mice (under mild ether anesthesia) are
collected at day 0 (pre-immune sera, 3 h before immunization), 14 days and
42 days (terminal samples) of priming.
(3)
Terminal blood samples are taken by cardiac puncture post euthanasia.
(4)
The serum is separated by centrifugation at 1500×
g
for 5 min after coagula-
tion for 30 min at room temperature.
(5)
Serum samples are stored at −22 °C until use.
(6)
The spleen of three mice from each group are surgically removed for the
cytokine assay.
7.4.2.5 Measurement of Humoral Response
The immune response in terms of level of OVA-specific IgG antibodies and
isotypes is established using indirect immunoassay. The assay is performed in
a 96-well plate.
