Biomedical Engineering Reference
In-Depth Information
(3)
Purify the antibody-modified IOMNPs, Ab-IOMNP, through magnetic
separation.
(4)
Wash the Ab-IOMNP two to three times with 10 mM borate buffer,
pH 7.4.
(5)
Establish the concentration of the IOMNPs in the Ab-IOMNP by measur-
ing the absorbance at 500 nm (subtract the background at 800 nm) against
a standard IOMNP.
(6)
To establish the concentration of the Ab in the Ab-IOMNP:
(a)
Take 1 µg of the Ab-IOMNP and add to 200 µg of anti-Ab-HRP.
(b)
Incubate for 30-60 min with gentle shaking.
(c)
Purify by magnetic separation.
(d)
Wash thoroughly (four to five times) with 10 mM borate buffer, pH 7.4.
(e)
Reconstitute with borate buffer.
(f)
Place in a 96-well plate and add 100 µL of TMB.
(g)
Cover the plate and incubate for 5 min.
(h)
Quench the reaction with 2 M H
2
SO
4
.
(i)
Record the absorbance at 450 nm.
(j)
Prepare a calibration standard by using various concentrations of the
anti-Ab-HRP.
(k)
Calculate the concentration of the Ab in Ab-IOMNP using the slope
of the anti-Ab-HRP curve.
(7)
Lyse the cells (10,000-100,000 cells in 100 µL 10 mM PBS, pH 7.4)
with trypsin or lysozyme for 30 min to release the cell membrane surface
proteins.
(8)
Add the lysed cells to 100 µL of 5 mg/mL of Ab-IOMNP.
(9)
Incubate for 1-2 h with gentle shaking.
(10)
Magnetize the proteins-Ab-IOMNPs for 1-2 h.
(11)
Wash the proteins-Ab-IOMNPs three times with 10 mM borate buffer,
pH 7.4
(12)
Expose the proteins-Ab-IOMNPs to 100 µL of at least 2 µg/mL HRP-
anti-proteins (the concentrations need to be optimized for optimum sig-
nals) for 1-2 h with gentle shaking.
(13)
Wash the HRP-anti-proteins-Ab-IOMNPs three times with 10 mM borate
buffer, pH 7.4.
(14)
Place 25 µL of the proteins-Ab-IOMNPs in 96-well plate.
(15)
Add 50-100 µL TMB.
(16)
Incubate for 5 min.
(17)
Remove the proteins-Ab-IOMNPs by magnetic separation for 10-15 min
resulting in a total incubation with the TMB for 20 min.
(18)
Transfer the TMB into a fresh plate.
(19)
Quench the reaction with 2 M H
2
SO
4
.
(20)
Record the absorbance at 450 nm.
(21)
Calculate the concentration of HRP-anti-protein HRP using a calibration
curve prepared as that of the anti-Ab-HRP.
