Biomedical Engineering Reference
In-Depth Information
(1)
Coat each well (NUNC-Immune Plate
®
Fb96 Maxisorb, Nunc, India) with
100 uL of antigen (100 µg/mL, 50 µg/mL, 25 µg/mL OVA in bicarbonate
buffer, pH 9.6).
(2)
Seal the plate and incubate at 4 °C overnight.
(3)
Wash the plate three times with PBS-Tween 20 (PBS-T, 0.05%, v/v).
(4)
Block with 150 µL of 3% gelatin in PBS for 90 min at 37 °C.
(5)
Wash three times with PBS-T.
(6)
Place 100 µL of serum (1:800) in gelatin in PBS-T20.
(7)
Incubate at RT for 90 min and wash again three times.
(8)
Put 100 µL of horse radish peroxidase (HRP) labeled goat anti-mouse
IgG, IgG2a or IgG1 (1:1000) in each well.
(9)
Seal the plates and incubate for 1 h at RT.
(10)
Repeat the washing step.
(11)
Place 100 µL of substrate O-phenylenediamine dihydrochloride (OPD)
(prepared in citrate buffer + H
2
O
2
) or tetramethyl benzidene (TMB)
enzyme substrate.
(12)
Incubate for at least 10 min at RT or until color development ensues.
(13)
When full color is developed, add 25 µL of 2.5 M H
2
SO
4
to stop the reaction.
(14)
Measure the absorbance at 492 nm. The OD at 492 or at 450 is a measure
of the level of immune response. The response is directly proportional
to the level of the IgG, IgG2, or IgG1 when the horse radish peroxidase
(HRP) is attached to goat anti-mouse IgG, IgG2a or IgG1, respectively.
A calibration curve for each of these antibodies must be established to
quantify the signals.
7.4.2.6 Estimation of Cytokine Levels
The effect of the OVA and the NMs were evaluated by Pandey
111
in terms of the
levels of IL-4 and IFN-γ in mouse spleen homogenates using ELISA kits for
cytokines.
116
This process involved the following protocol.
(1)
Separate and weigh the individual spleens from euthanized mice.
(2)
Homogenize the spleens in ice-cold RPMI containing 1% 3-(cholamido-
propyl-
o
-dimethylammonio)-1-propanesulphonate (CHAPS; Sigma) in a
micro tissue homogenizer.
(3)
Store the 10% (w/v) homogenates for 1 h at 2-4 °C.
(4)
Centrifuge at 2000×
g
for 20 min to remove insoluble debris.
(5)
Apply the mouse cytokine assay kit from eBioscience (San Diego, CA) to
determine the levels of cytokines following the manufacturer's instructions.
(6)
Use the standard curve provided by the manufacturer for IL-4 and IFN-γ to
estimate the levels that are expressed in pg/mL.
Fifteen days after therapy, animals were challenged with OVA and different signs
of anaphylactic shock were evaluated. Developed aquasomes possessed a nega-
tive zeta potential (−11.3 mV) and an average size of 47 nm with OVA adsorption
efficiency of ~60.2 µg mg
−1
of hydroxyapatite core. In vivo immune response
