43
Figure 4: Sensorgram of sequential binding of antibody, antigen and secondary antibody. The antigen was Influenza A nucleoprotein (InfA NP) at 10 µg mL-1
in TBS-T. Antibodies
Figure 2. Schematic representations of IgG-binding domains fused to OmpA with bound antibody or Fab fragment. The dimensions shown were measured by polarised neutron reflection [6]. The total height of the capture layer and the antibody was measured at close to 190 Å suggesting that the antibody is not completely upright as depicted but is partially tilted towards the surface. Consequently, the distance between the antigen binding site and the gold surface is only ~20nm and this is a great advantage for most types of biosensor.
InA 245 (mouse mAb IgG2b) and InA 108 (mouse mAb IgG1) were obtained from Hytest, Finland, and were diluted to 30 µg mL-1
in TBS-T. ORLA18 surface was pre-assembled in
Fc1 and ORLA87 surface in Fc2 both with PEG-thioalkane filler. TBS-T washes were carried out between injections. A control run without antigen was also carried out.
The surfaces were regenerated with 100 mM HCl after each cycle. The flow rate was 5 µL min-1
throughout. A
In a second experiment (on a Biacore X-100 machine) the binding of antibody was followed by the binding of antigen and a secondary antibody that binds at a site different from the primary antibody for example, to mimic a typical ‘sandwich’ immunoassay (Figure 4). The molar ratio of antigen to antibody was calculated (Table 3). A change of +1 RU was taken to represent 1pg/mm2
of protein [7]. The
molecular weight of InA245 is 150 kDa and that of the NP protein is 57 kDa. The molar ratio of 2.1 shows that there are two molecules of antigen binding for every one of antibody for example, 100% of the antibody binding sites are available for antigen binding. This compares to 25-50% for amine coupled antibody and 1-5% for adsorbed antibody; clearly demonstrating the advantage of correctly oriented antibody. The sandwich assay was carried out using a range of antigen concentrations. A plot of the change in signal upon secondary antibody addition is shown in Figure 5. These data show the usefulness of the antibody capture monolayer in ‘on-chip’ immunoassay applications.
Table 3. Molar ratio of antigen to antibody. ORLA18
Relative nmol/mm2 Response
InA 245 B
1° Antibody Antigen
InfA NP
Molar ratio Ag:1°Ab
2.1 2.1 416.6 327.1 2.8 x 10-6 5.8 x 10-6
ORLA87
Relative nmol/mm2 Response
1479.2 1 x 10-5 1202.3 2.1 x 10-5
Surface stability and longevity
The stability of ORLA18 surface over 30 cycles of antibody injection and regeneration was tested by SPR on Biacore (Figure 6). There was a net loss of binding signal response of 10% over 30 cycles. The quantity of antibody bound at the last cycle was greatly in excess of that required for antigen detection.
C
The longevity of the assembled surface was tested using surface acoustic wave (SAW) technology. ORLA18 protein surfaces with a PEG-thioalkane filler were assembled on the gold surface of SAW chips (Japan Radio Co. Ltd) and the binding of IgG analysed over 6 cycles. The chips were regenerated with 100 mM HCl and dried in nitrogen and stored in airtight containers at 4°C. The binding of IgG was analysed again after 144 days (~20 weeks) of storage. The average phase angle change on day 1 was -52.79° (± 2.33) whereas that on Day 144 was -48.42° (± 2.25) indicating that the surfaces are very stable.
Figure 3. Sensorgrams showing binding of rabbit polyclonal IgG (A), mouse monoclonal
IgG1 (B) or mouse monoclonal IgG2a (C) analysed by surface plasmon resonance. Protein-PEG thioalkane monolayers were assembled in situ on a Biacore Au chip. Antibody was diluted in tris-buffered saline, 0.05% Tween 20 (TBS-T) at 30 µg mL-1
flow rate of 5 µL min-1 at a
. Antibody injection was started at 80s for 6 min until 440s. Then TBS-T was flowed over the surface for approx 13 min. Surfaces were regenerated with 100 mM HCl for 3 min prior to next antibody injection.
Figure 5. Sensorgram showing the concentration of InfA NP Vs the response obtained after secondary antibody injection on Biacore. The lower detection limit was 10-100 ng/mL on ORLA18 and 100 ng/mL on ORLA87.
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52
