Performance specifications – Bio-Rad ProteinChip Antibody Capture Kit User Manual

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Covalently Crosslinking IgG Antibodies to Protein G

Antibodies captured by protein G on ProteinChip PG20 arrays are not covalently
bound to the surface of the array. When these arrays are analyzed in a ProteinChip
SELDI reader, peaks may be seen in the resulting spectra corresponding to intact
IgG (MH+ ~148 kD, M2H+ ~75 kD) or its fragments (~75 kD, ~48 kD, ~22 kD),
especially if a high laser energy is used. These peaks may interfere with the detection
or quantitation of an antigen of similar molecular weight. When the antibody is
crosslinked to protein G on the array, it becomes covalently attached to the array
surface and will not be desorbed from the array surface during the reading process.

The results of an antibody capture assay depicting crosslinked and noncrosslinked
antibody are shown in Figure 3. Without crosslinking, an IgG peak (molecular mass
148 kD) is often seen. When the IgG is crosslinked to the ProteinChip PG20 array,
the IgG peak is not observed. Crosslinking does not affect the peak intensity of
TNF-

α antigen.

Fig. 3. Effect of crosslinking on TNF-

α antigen and TNF-α antibody peak profiles. A, peak

corresponding to the 17.5 kD protein, TNF-

α, is seen when 100 fmol TNF-α is applied to a ProteinChip

PG20 array; B, peak corresponding to the 145 kD TNF-

α IgG antibody is seen when 2.7 pmol of antibody is

applied to a ProteinChip PG20 array; C, peak corresponding to the 17.5 kD protein, TNF-

α, is seen

when 100 fmol TNF-

α is applied to a ProteinChip PG20 array and the anti-TNF-α antibody is crosslinked to

the array; D, no peak is seen corresponding to the 145 kD TNF-

α IgG antibody when 2.7 pmol of antibody

is added to a ProteinChip PG20 array.

Performance Specifications

Using the reagents supplied with the ProteinChip antibody capture kit, the user
should be able to detect the TNF-

α antigen positive control (1.0 µM recombinant

human TNF-

α antigen in 0.1% BSA/PBS) down to a level of 1 fmol at a signal-to-

noise ratio of at least 3. For the TNF-

α control, the quantitation range will generally

be from 10–500 fmol.

Different antigens behave significantly differently in the antibody capture and SELDI
processes, so these values cannot be applied to any other antigen-antibody
system. The lower limit of detection and dynamic range for each antigen needs to
be determined for each system.

Not Crosslinked

Crosslinked

.75

.50

.25

.075

.050

.025

15,000

17,500

20,000

120,000

140,000

160,000