Biology Reference
In-Depth Information
3.2.2. Determination of Serum Anti-SLO Antibody Concentrations
Three-milliliter blood samples are taken on day minus-seven of the protocol
to enable determination of serum anti-SLO antibody titers (
see
Table 1
). The
sera isolated will also serve as a pre-infection control for antibody measure-
ments comparing pre- and post-infection samples. Blood samples are isolated
and processed as described in
Subheading 3.4.1
. Sera samples isolated from
the macaques are titered for anti-SLO antibody concentrations by ELISA as
follows:
1. Add 100 μL of SLO solution [2.5 μg of Sigma-Aldrich SLO (contains 3-9%
SLO protein) per milliliter of PBS] to the wells of a 96-well ELISA plate. Dry
overnight at 40 °C.
2. Wash wells with PBST.
3. To each well add 100 μL of 1% BSA in PBST and incubate on a rocking
platform for 30 min at room temperature to block.
4. Wash wells with PBST.
5. To triplicate wells add 100 μL of each serum sample (diluted 1:500 with PBS)
and incubate for2hatroom temperature on a rocking platform.
6. Wash wells with PBST.
Table 1
Timeline of Sample Isolations During the Non-Human Primate Experiment
Day of the experiment Clinical
scoring
Throat swab
Blood
withdrawal
Nasal wash
Saliva
-7 Monday
X
X
X
0 Monday
X
X
X
X
2 Wednesday
X
X
4 Friday
X
X
7 Monday
X
X
X
X
X
10 Thursday
X
X
14 Monday
X
X
X
X
X
17 Thursday
X
X
21 Monday
X
X
X
X
X
28 Monday
X
X
X
X
X
Throat swabs, blood withdrawals, nasal washes and saliva samples are isolated on the
indicated (X) days. Blood specimens (3 mL) are taken by venipuncture from the cephalic vein.
Nasal washes are collected by instilling 1.5 mL of sterile phosphate-buffered saline (PBS) into
the nostril and immediately aspirating wash fluid and secretions with a sterile syringe. Saliva
specimens are collected by aspiration from the cheek pouch. Prior to throat swabbing, clinical
scoring of tonsillitis and pharyngeal erythema severity is performed by a trained veterinarian
who is blinded to the two animal groups.
Search WWH ::
Custom Search