Biomedical Engineering Reference
In-Depth Information
(11)
Wash three times with buffer D.
(12)
Take 20-100 µL of the IOMNP-GAM and add to 980-900 µL buffer D.
(13)
Take the absorbance at 800 and at 500 nm. Calculate the concentration of
the IOMNP using the equation:
mg
mL
=
A
500
−
A
800
X
where
A = absorbance and X = the factor that is a function of IOMNP diameter
which is equivalent to 5 for 15-50 nm and 4 for <15-nm diameter.
(14)
Reconstitute with buffer D to 1 mg/mL IOMNP-GAM or as desired.
(15)
Run the original IOMNP-COOH and the IOMNP-GAM in 1-1.5% aga-
rose gel at 100V for 30 to 40 min to verify conjugation efficiency.
(16)
Place 25 µL of the 1 mg/mL IOMNP-GAM in wells of a 96-well plate.
B. Quantification of the number of Ab on the NP surface
(17)
Add 20 µg/mL of DAG-HRP at 0, 1, 5, 10, and 25 µL to the wells contain-
ing the IOMNP-GAM. Add buffer D to make a total volume of 50 µL in
each of the wells. Do this in triplicate.
(18)
Incubate for 30 min at RT with shaking to form the IOMNP-GAM +
DAG-HRP complex.
(19)
Purify the IOMNP-GAM + DAG-HRP by ultracentrifugation or by mag-
netic separation.
(20)
Wash the IOMNP-GAM + DAG-HRP with buffer D two times.
(21)
Incubate the IOMNP-GAM + DAG-HRP with blocking buffer for 10 min
at RT.
(22)
Wash the IOMNP-GAM + DAG-HRP with buffer D two times.
(23)
Reconstitute the IOMNP-GAM + DAG-HRP with 25 µL of buffer D.
(24)
Add 50 µL of TMB to each of the wells and gently shake for 5 min.
(25)
Add 25 µL of 2 N HCl to quench the reaction.
(26)
Magnetize and aspirate the supernatant into clean wells on the 96-well plate.
(27)
Read the absorbance at 450 nm.
(28)
Compare the results against a calibration standard.
C. Preparation of the calibration standard
(1)
Place 50 µL of TMB in wells of a 96-well plate.
(2)
Add 24.8, 24.5, 24.25, 24, 23, 21, and 25 µL of buffer D.
(3)
Add 0, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, and 10 µL of DAG-HRP at 10 µg/mL.
(4)
Incubate for 5 min with gentle shaking at RT.
(5)
Add 25 µL of 2 N HCl to quench the reaction.
(6)
Read the absorbance at 450 nm.
(7)
All studies must be at least in triplicate to be statistically significant.
(8)
Graph the signal versus the concentration of DAG-HRP to generate the
calibration standard.
