Protein Orientation and Subsequent Activity Optimised Antibody Immobilisation Protocol

The immobilisation of a mouse IgG3 antibody raised to Cortisol (anticortisol) is summarised in Table 3. The experimental protocol was as follows. A thiol functionalised chip was first calibrated using standard calibrant solutions and then subjected to N-maleimidobutyryloxy-sulpho-succinimide ester (sulpho-GMBS), which is a thiol amine linker followed by protein G. Once the protein G had been immobilised (via the sulpho-GMBS linker) the surface was ready for the immobilisation of the IgG3 antibody itself. Protein G has the capability to orient the antibody by interacting specifically with the Fc region, ensuring that the antibody remains active once immobilised. As can be seen from Table 3, the layer thickness increases by over 15 nm on the addition of the antibody. This is entirely consistent with the long axis of the IgG3 antibody (typically 12-15 nm, the other two axes typically being of the order of 5-10 nm) being immobilised normal to the sensor surface. This is shown schematically in Fig. 5.

Table 3. Layer analysis for each layer of the oriented immobilisation protocol for the IgG3 anticortisol antibody. GMBS N-(y-maleimidobutyryloxy)succinimide

Layer Name Thickness (nm) Density (g cm-3) Mass (ngmm-2)

1 GMBS 0.540 0.555 0.300

2 Protein G 0.826 0.810 0.669

3 IgG3 anti-cortisol 15.126 0.181 2.743

Fig. 5. Schematic diagram of the immobilisation of IgG3 anticortisol to the sensor chip surface. GMBS N-(y-maleimidobutyryloxy)succinimide

Having prepared the immobilised antibody surface, it was repeatedly challenged with the antigen, cortisol. This is an extremely small molecule (molecular mass 372 Da) when compared to the antibody (molecular mass 160,000 Da). This situation is germane to many systems of interest to the life scientist and provides a considerable challenge for instrumentation scientists. As the size and mass of the protein, in this case an antibody, increases and the size and mass of the molecule that binds, in this case cortisol, decreases, so the signal to noise ratio diminishes. However, the sensitivity of DPI can be seen, from Fig. 6, to be entirely adequate to monitor the antigen-antibody binding event on injecting antigen across the immobilised antibody surface. During repeated injections of cortisol and subsequent washes, the thickness of the antibody layer increases and its density reduces on binding (which is reversed during the wash), which is indicative of a structural change occurring as a consequence of the binding process.

These structural changes occur despite the considerable difference in size between the antibody and antigen, the antibody surface undergoing a substantial structural change on binding the antigen. Each time the antibody surface is subjected to the antigen, an increase in the thickness of the layer (up to 0.5 nm at high antigen concentrations) and a commensurate decrease in density is observed. It should be noted that despite the density decrease, the overall layer mass increases on binding antigen. The relationship between the thickness and density of immobilised protein layers provides very strong evidence of specific binding events (Swann et al. 2004).

Fig.6. Structural responses when anti-cortisol binds Cortisol (at varying Cortisol concentrations)

Both the mass increase and thickness increase can be used to determine the affinity constant for the binding interaction and in this case for the thickness change binding curve (see Fig. 7). These observations are consistent with the two flexible FAB arms becoming more erect on binding antigen. From the mass values it was also possible to calculate the stoichiometry for the binding event, which at two antigen molecules per antibody is as would be expected for an IgG3 antibody. As the binding was repeated at a range of antigen concentrations it was also possible to calculate an affinity constant for the antigen binding. The value of 0.8 ^M was obtained, which is within the range expected by the supplier of the antibody, strongly supporting the hypothesis that the observed structural changes were directly related to antibody-antigen binding events.

Poorly Optimised Antibody Immobilisation

This example is provided to demonstrate how the technique can be used to determine the orientation and structure of an immobilised antibody layer and subsequent blocking steps and to diagnose potential problems at an early stage. In this case, anti-ovalbumin has been immobilised to the sensor surface using the homobifunctional linker bis(sulphosuccinimydyl) suberate (BS3). It can be seen that in this case the antibody goes down as a very thin layer and is therefore likely to be prone on the surface (Table 4). In such a condition, the antibody cannot function correctly and is likely to have a very low activity. This indeed was observed to be the case. The experiment was, however, continued and a blocking step was undertaken. It can be seen

Fig. 7. Structural binding curve for the binding of cortisol to anti-cortisol

Table 4. Layer structure during the immobilised of anti-ovalbumin. TM Transverse magnetic, TE transverse electric, Diff phase difference, Th thickness, RI refractive index, BS3 bis(sulphosuccinimydyl) suberate, Ab antibody

Name Mass (ng mm-2) TMDiff(rad) TEDiff(rad) Th (nm)

BS3 Ab

0.255 0.720

0.827 1.526

0.693 1.151

0.486 3.334

1.4303 1.3731

Table 5. Addition of blocking agent and antigen to the anti-ovalbumin surface

Name Mass (ng mm-2) TMDiff(rad) TEDiff(rad) Th (nm) RI

Block 1.406 2.179 1.715 6.117 1.3757

Antigen 1.446 0.121 0.094 6.403 1.3749

from Table 5 that the addition of a block, y-globulin, simply increases the thickness of the layer present but does not increase its refractive index (and therefore its density). This suggests that the blocker in the experiment is not inserting between the layers as anticipated, but simply covering the surface of the prone antibody layer. Finally, when the antigen is added to the layer structure, this too appears to add additional layer thickness without increasing the layer density, suggesting that the antigen can only interact with the surface generated by the blocker.

Table 6. Example conditions and immobilisation strategies used with antibodies. MW Molecular weight, HSA human serum albumin, +ve positive, -ve negative, ProBNP precursor of brain natriuretic peptide

Antibody Type Immobilisation Antibody Activity Antigen AThickness

Table 6. Example conditions and immobilisation strategies used with antibodies. MW Molecular weight, HSA human serum albumin, +ve positive, -ve negative, ProBNP precursor of brain natriuretic peptide

Antibody Type Immobilisation Antibody Activity Antigen AThickness

Layer (nm)













Active «1:1





-nh2,sa, s-NHS-LC Biotin


Active 1:1





-NH2,BS3, Protein G





Cortisol (1)

Protein G


Active 2:1



Cortisol (2)


-SH, s-GMBS,




Protein G 2:1

Using Structural Information to Optimise Antibody Activity

An example range of immobilisation strategies and antibody classes is shown in Table 6. It can be seen that active antibody surfaces can be obtained with a wide range of immobilisation strategies.

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  • Aleena Stevenson
    How to optimise antibody orientation?
    3 years ago

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