Characterization of Monoclonal Antibody Epitope Specificity Using Biacore’S SPR Technology

APPLICATION NOTE 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 surface- attached 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 deter- SPR response is measured in resonance mination is the same as that described units (RU). For most proteins, 1000 RU for RIA- or ELISA-based techniques, but corresponds to a surface concentration of the use of SPR offers several important approximately 1 ng/mm2 [9]. advantages: Immobilization of RAMG1 on the • None of the interacting components sensor chip needs to be purified or labelled in any RAMG1 was covalently coupled to a way. As a result, the mapping can be Sensor Chip CM5 via primary amine performed using small amounts of groups using the conditions listed in Table unfractionated MAbs in cell culture 1. The resulting sensorgram (Figure 1) supernatants. shows that RAMG1 corresponding to • A mass-dependent SPR response is about 12000 RU is covalently linked to the obtained from the binding of each sensor chip surface. component to the sensor surface [9]. All stages in the binding process can Pair-wise binding of MAbs thus be monitored. Pair-wise binding of MAbs to p24 was • Each stage of the binding sequence is tested using the conditions shown in Table easily quantified, aiding the interpret- 2. Each analysis cycle concludes with ation of the results. removal of all non-covalently bound • The technique allows multi-site material from the sensor chip surface, specificity tests using a sequence of regenerating the surface in preparation for several MAbs. a new cycle. One cycle takes approximately • The average assay time is short (15 15 minutes to perform and in this example, minutes), and large numbers of analyses 60 cycles were run automatically. can be processed 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: 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Surfactant P20 NHS: 100 mM N-hydroxysuccinimide in

H2O EDC: 400 mM 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide in H2O RAMG1: RAMG1, 30 µg/ml in 10 mM Na-acetate pH 5.0 Ethanolamine: 1 M ethanolamine hydrochloride, adjusted to pH 8.5 Table 1 with NaOH Procedure for immobilizing RAMG1 on a Sensor Chip HCl: 100 mM HCl CM5, to make a specific surface for adsorption of MAbs from hybridoma supernatants. Biacore immobilization protocol Buffer flow is maintained at 0 min HBS-EP, flow 5 µl/min Start cycle 5 µl/min throughout the immobilization protocol. 5 min Mix NHS + EDC 1:1 Activate surface Inject 30 µl 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. Reagents HBS-EP buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Surfactant P20 Salt-free HBS: HBS with NaCl omitted First MAb: Undiluted hybridoma supernatant containing first MAb Blocking Ab: α-AFP, 50 µg/ml in salt-free HBS p24: p24, 10 µg/ml in 10 mM Na-acetate, pH 5.0 Table 2 Procedure for testing Second MAb: Undiluted hybridoma supernatant containing second simultaneous binding of MAb two MAbs to p24. Buffer flow HCl: 100 mM HCl is maintained at 5 µl/min throughout the analysis protocol. 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 tests were run only when a negative result pair-wise epitope specificity studies. The was obtained, to ensure that the absence of MAbs tested show simultaneous binding, binding was not an artefact of the sequence and are therefore judged to bind to of attachment. The final complete mapping independent epitopes. analyzed 537 binding tests, of which 185 It is essential that unoccupied RAMG1 were reciprocal duplicates with the same sites on the sensor chip surface are blocked antibodies in reversed order. before injection of the second MAb super- Four of the MAbs gave negative results natant, to avoid false positive responses. when used as the first antibody, regardless This is assured by using a concentration of of which MAb was tested as the second blocking antibody sufficient to saturate the antibody. Closer examination of the surface even in the absence of the first sensorgrams showed that these MAbs lost MAb. Although different first MAbs the ability to bind antigen when they were bound to different extents, the SPR signal attached to the surface through RAMG1, level reached after injection of the blocking although positive binding was seen in MAb was the same regardless of the many cases when these MAbs were used as amount of first MAb bound. This confirms second antibody. that the first MAb and blocking antibody These observations illustrate two together occupy all the available sites. particularly valuable features of Biacore’s Two kinds of control experiment ensure SPR technology in comparison with other that the second MAb binds to the antigen epitope mapping techniques: the reason for and not to the RAMG1 or another the negative response (lack of antigen component on the sensor chip surface: binding) is directly apparent from the • Omission of p24 from the normal assay sensorgram, and reversed-order pair-wise sequence reduces the response from the tests are easily performed. second MAb supernatant to background The reactivity patterns for the MAbs tested levels. For each supernatant, the mean are shown as a 30x30 matrix in Figure 3. background obtained with four arbitrarily chosen first MAbs was subtracted from all responses (typical background levels are 30-100 RU). • Binding of both purified MAb and p24 is eliminated if blocking antibody is injected before the first MAb. This also shows that exchange between surface- bound blocking antibody and MAb in free solution is negligible on the time scale of one assay cycle. Figure 3 In all, the epitope specificity of 30 different Reactivity pattern matrix showing the binding MAbs was characterized. Theoretically, ability of pairs of MAbs this requires 900 tests for the complete to p24. 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 Grouping MAbs that show the same reactivity pattern gives 17 groups representing epitopes (Figure 4), which Figure 4 may be visualized in a two-dimensional Grouping 30 MAbs according to their ‘‘surface-like’’ map shown in Figure 5. reactivity patterns Note that the diagram does not necessarily identifies 17 proposed epitope regions. 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 multi- Figure 5 determinant test is shown in Figure 6. Two-dimensional Here, with p24 linked to the surface ‘‘surface-like’’ map of the epitopes based on through MAb 31, MAbs 41 and 44 are the matrix in Figure 4. both prevented from binding, while MAbs Overlapping circles represent MAb groups 17, 33, 23, 5 all bind independently of within which pairs of each other in that order. The last antibody, MAbs cannot bind simultaneously. 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 Figure 6 tests, until a plateau was reached in the Multi-determinant SPR signal. binding of MAbs to p24. The MAbs injected at each stage are identified with reference to the diagram obtained from two-site specificity studies. Results

The work in this Application Note binding step in real time is automatically demonstrates that Biacore’s SPR recorded, so that both kinetic and technology can be used to characterize equilibrium parameters may be assessed epitope specificity with MAbs in for macromolecular interactions. The unfractionated hybridoma culture technique is well suited to programmed supernatant. The quantitative data operation, and can handle many samples obtained for each step in the binding without user intervention. This feature is process permits a more comprehensive interpretation of the binding than is important in epitope specificity possible with conventional techniques. determination of a large panel of MAbs, Although this study concerned only levels where the pair-wise combination matrix of antibody binding, the progress of each requires a large number of assay cycles. References

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