A P P L I C A T I O N N O T E 1 Characterization of monoclonal antibody epitope specificity using Biacore s SPR technology Abstract Biacore s SPR technology based on surface plasmon resonance technology has been used to map the epitope specificity patterns of 30 monoclonal antibodies against recombinant HIV-1 core protein p24. The technique does not require labelling of either antibodies or antigen, and all specificity determinations were performed with antibodies in unfractionated hybridoma culture supernatants. Pair-wise binding tests divided the 30 antibodies into 17 groups, representing 17 epitopes on the antigen. Introduction Epitope mapping using monoclonal antibodies (MAbs) is a powerful tool in examining the surface topography of macromolecules. Through its binding, each MAb defines one specific site, or epitope, on the antigen, and a pair of MAbs which bind to closely situated epitopes will interfere sterically with each other s binding [1,2,3]. Determination of epitope specificity is an important part of MAb characterization for both investigative work and medical and industrial applications. The epitope specificity of a panel of MAbs is most easily determined by testing the ability of pairs of MAbs to bind simultaneously to the antigen. MAbs directed against separate epitopes will bind independently of each other, whereas MAbs directed against closely related epitopes will interfere with each other s binding. The most common technique for determining epitope specificities tests pair-wise binding with RIA or ELISA [4]. One antibody is attached to a solid substrate, the antigen is bound, and the ability of the second antibody to bind to the surfaceattached complex is tested. A drawback with these methods is that the secondary interactant must be labelled in some way. Simultaneous binding, indicating distinct epitopes, is readily identified, but it is generally more difficult to interpret an absence of simultaneous binding. This Application Note describes the characterization of epitope specificity patterns of 30 different MAbs directed against recombinant HIV-1 core protein p24. Biacore s SPR technology [5,6] based on surface plasmon resonance (SPR) [7,8] is used to measure binding of macromolecular components to each other at a sensor chip surface.
The principle of the specificity determination is the same as that described for RIA- or ELISA-based techniques, but the use of SPR offers several important advantages: None of the interacting components needs to be purified or labelled in any way. As a result, the mapping can be performed using small amounts of unfractionated MAbs in cell culture supernatants. A mass-dependent SPR response is obtained from the binding of each component to the sensor surface [9]. All stages in the binding process can thus be monitored. Each stage of the binding sequence is easily quantified, aiding the interpretation of the results. The technique allows multi-site specificity tests using a sequence of several MAbs. The average assay time is short (15 minutes), and large numbers of analyses can be processed automatically. SPR response is measured in resonance units (RU). For most proteins, 1000 RU corresponds to a surface concentration of approximately 1 ng/mm 2 [9]. Immobilization of RAMG1 on the sensor chip RAMG1 was covalently coupled to a Sensor Chip CM5 via primary amine groups using the conditions listed in Table 1. The resulting sensorgram (Figure 1) shows that RAMG1 corresponding to about 12000 RU is covalently linked to the sensor chip surface. Pair-wise binding of MAbs Pair-wise binding of MAbs to p24 was tested using the conditions shown in Table 2. Each analysis cycle concludes with removal of all non-covalently bound material from the sensor chip surface, regenerating the surface in preparation for a new cycle. One cycle takes approximately 15 minutes to perform and in this example, 60 cycles were run automatically. Materials and methods Materials SPR measurements were performed using a Biacore system. Sensor Chip CM5 and Amine Coupling Kit for immobilization were from Biacore AB. Immunosorbent purified rabbit anti-mouse IgG1 (RAMG1), hybridoma culture supernatants containing murine MAbs against recombinant HIV-1 p24, and monoclonal anti-human alpha-fetoprotein (a-afp) were obtained from Pharmacia Diagnostics AB, Uppsala. Recombinant HIV-1 core protein p24 was supplied by Pharmacia Genetic Engineering Inc., San Diego.
Reagents HBS-EP buffer: NHS: EDC: 10 mm HEPES ph 7.4, 150 mm NaCl, 3.4 mm EDTA, 0.005% Surfactant P20 100 mm N-hydroxysuccinimide in H 2O 400 mm 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in H 2O RAMG1: RAMG1, 30 µg/ml in 10 mm Na-acetate ph 5.0 Ethanolamine: 1 M ethanolamine hydrochloride, adjusted to ph 8.5 with NaOH HCl: 100 mm HCl Biacore immobilization protocol 0 min HBS-EP, flow 5 µl/min Start cycle 5 min Mix NHS + EDC 1:1 Activate surface Inject 30 µl Table 1 Procedure for immobilizing RAMG1 on a Sensor Chip CM5, to make a specific surface for adsorption of MAbs from hybridoma supernatants. Buffer flow is maintained at 5 µl/min throughout the immobilization protocol. 11 min Inject 30 µl RAMG1 Couple RAMG1 19 min Inject 30 µl ethanolamine Deactivate excess reactive groups 26 min Inject 15 µl HCl Remove non-covalently bound material 30 min End cycle Figure 1 Sensorgram obtained from immobilization of RAMG1 on a Sensor Chip CM5. Numbers on the sensorgram indicate injections as follows: (1) NHS/EDC, (2) RAMG1, (3) ethanolamine, (4) HCl. Note that the SPR signal is off scale at the top of the RAMG1 peak, while the RAMG1 solution is in contact with the sensor chip.
Table 2 Procedure for testing simultaneous binding of two MAbs to p24. Buffer flow is maintained at 5 µl/min throughout the analysis protocol. Reagents HBS-EP buffer: Salt-free HBS: First MAb: Blocking Ab: Second MAb: HCl: 10 mm HEPES ph 7.4, 150 mm NaCl, 3.4 mm EDTA, 0.005% Surfactant P20 HBS with NaCl omitted Undiluted hybridoma supernatant containing first MAb α-afp, 50 µg/ml in salt-free HBS p24: p24, 10 µg/ml in 10 mm Na-acetate, ph 5.0 Undiluted hybridoma supernatant containing second MAb 100 mm HCl Biacore analysis protocol 0 min HBS-EP, flow 5 µl/min Start cycle 1 min Inject 4 µl first MAb Bind to RAMG1 4 min Inject 4 µl blocking Ab Block free RAMG1 sites 7 min Inject 4 µl p24 Bind antigen to first MAb 9.5 min Inject 4 µl second MAb Test binding 13 min Inject 10 µl HCl Regenerate surface 15 min End cycle Figure 2 Example of a sensorgram obtained from epitope specificity determination for two MAbs directed against independent epitopes. The SPR response gives the amount of surface-bound component at each stage as follows: (A) baseline signal, (B)-(A) first MAb, (C)-(B) blocking antibody, (D)-(C) p24, (E)-(D) second MAb.
Results Figure 2 shows a typical sensorgram from pair-wise epitope specificity studies. The MAbs tested show simultaneous binding, and are therefore judged to bind to independent epitopes. It is essential that unoccupied RAMG1 sites on the sensor chip surface are blocked before injection of the second MAb supernatant, to avoid false positive responses. This is assured by using a concentration of blocking antibody sufficient to saturate the surface even in the absence of the first MAb. Although different first MAbs bound to different extents, the SPR signal level reached after injection of the blocking MAb was the same regardless of the amount of first MAb bound. This confirms that the first MAb and blocking antibody together occupy all the available sites. Two kinds of control experiment ensure that the second MAb binds to the antigen and not to the RAMG1 or another component on the sensor chip surface: Omission of p24 from the normal assay sequence reduces the response from the second MAb supernatant to background levels. For each supernatant, the mean background obtained with four arbitrarily chosen first MAbs was subtracted from all responses (typical background levels are 30-100 RU). tests were run only when a negative result was obtained, to ensure that the absence of binding was not an artefact of the sequence of attachment. The final complete mapping analyzed 537 binding tests, of which 185 were reciprocal duplicates with the same antibodies in reversed order. Four of the MAbs gave negative results when used as the first antibody, regardless of which MAb was tested as the second antibody. Closer examination of the sensorgrams showed that these MAbs lost the ability to bind antigen when they were attached to the surface through RAMG1, although positive binding was seen in many cases when these MAbs were used as second antibody. These observations illustrate two particularly valuable features of Biacore s SPR technology in comparison with other epitope mapping techniques: the reason for the negative response (lack of antigen binding) is directly apparent from the sensorgram, and reversed-order pair-wise tests are easily performed. The reactivity patterns for the MAbs tested are shown as a 30x30 matrix in Figure 3. Binding of both purified MAb and p24 is eliminated if blocking antibody is injected before the first MAb. This also shows that exchange between surfacebound blocking antibody and MAb in free solution is negligible on the time scale of one assay cycle. In all, the epitope specificity of 30 different MAbs was characterized. Theoretically, this requires 900 tests for the complete map if all pairs are to be tested in both binding sequences. In practice, however, many of the pairs will be redundant, since a positive result in the first sequence tested indicates distinct epitopes. Reciprocal pair Figure 3 Reactivity pattern matrix showing the binding ability of pairs of MAbs to p24.
Figure 4 Grouping 30 MAbs according to their reactivity patterns identifies 17 proposed epitope regions. Figure 5 Two-dimensional surface-like map of the epitopes based on the matrix in Figure 4. Overlapping circles represent MAb groups within which pairs of MAbs cannot bind simultaneously. Figure 6 Multi-determinant binding of MAbs to p24. The MAbs injected at each stage are identified with reference to the diagram obtained from two-site specificity studies. Grouping MAbs that show the same reactivity pattern gives 17 groups representing epitopes (Figure 4), which may be visualized in a two-dimensional surface-like map shown in Figure 5. Note that the diagram does not necessarily correspond to a physical map of the binding sites on the antigen surface, since conformational changes in the antigen or electrostatic interactions between MAbs may distort the binding patterns. In this particular case, however, the results do not contradict a simple two-dimensional surface-like interpretation of the map. Biacore s SPR technology can easily be applied to multi-determinant binding experiments, in addition to the simpler pair-wise binding tests. An example of a sequential multideterminant test is shown in Figure 6. Here, with p24 linked to the surface through MAb 31, MAbs 41 and 44 are both prevented from binding, while MAbs 17, 33, 23, 5 all bind independently of each other in that order. The last antibody, MAb 7, does not bind, as expected from the pair-wise exclusion of MAbs 5 and 7. These results accord well with the conclusions from the epitope specificity studies. Note that in this type of experiment, saturation of the surface binding sites at each stage is essential. Each MAb was therefore injected over a longer time period than for the pair-wise binding tests, until a plateau was reached in the SPR signal.
Results The work in this Application Note demonstrates that Biacore s SPR technology can be used to characterize epitope specificity with MAbs in unfractionated hybridoma culture supernatant. The quantitative data obtained for each step in the binding process permits a more comprehensive interpretation of the binding than is possible with conventional techniques. Although this study concerned only levels of antibody binding, the progress of each binding step in real time is automatically recorded, so that both kinetic and equilibrium parameters may be assessed for macromolecular interactions. The technique is well suited to programmed operation, and can handle many samples without user intervention. This feature is important in epitope specificity determination of a large panel of MAbs, where the pair-wise combination matrix requires a large number of assay cycles.
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