A P P L I C A T I O N N O T E 1

Characterization of monoclonalantibody epitope specificity using

Biacore’s SPR technology

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.

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.

Abstract

Introduction

The principle of the specificity deter-

mination 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 interpret-

ation 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.

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.

SPR response is measured in resonance

units (RU). For most proteins, 1000 RU

corresponds to a surface concentration of

approximately 1 ng/mm2 [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

Table 1Procedure for immobilizingRAMG1 on a Sensor ChipCM5, to make a specificsurface for adsorption of MAbsfrom hybridoma supernatants.Buffer flow is maintained at 5 µl/min throughout theimmobilization protocol.

Figure 1Sensorgram obtained fromimmobilization of RAMG1 on a Sensor Chip CM5. Numberson the sensorgram indicateinjections as follows: (1) NHS/EDC, (2) RAMG1, (3) ethanolamine, (4) HCl.

Note that the SPR signal is offscale at the top of the RAMG1peak, while the RAMG1solution is in contact with thesensor chip.

Reagents

HBS-EP buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mMEDTA, 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 8.5with 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 surfaceInject 30 µl

11 min Inject 30 µl RAMG1 Couple RAMG1

19 min Inject 30 µl ethanolamine Deactivate excessreactive groups

26 min Inject 15 µl HCl Remove non-covalently bound material

30 min –– End cycle

Table 2Procedure for testingsimultaneous binding oftwo MAbs to p24. Buffer flowis maintained at 5 µl/minthroughout the analysisprotocol.

Figure 2Example of a sensorgramobtained from epitopespecificity determination fortwo MAbs directed againstindependent epitopes. TheSPR response gives theamount of surface-boundcomponent at each stage asfollows: (A) baseline signal,(B)-(A) first MAb, (C)-(B)blocking antibody, (D)-(C) p24,(E)-(D) second MAb.

Reagents

HBS-EP buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mMEDTA, 0.005% Surfactant P20

Salt-free HBS: HBS with NaCl omitted

First MAb: Undiluted hybridoma supernatant containing firstMAb

Blocking Ab: α-AFP, 50 µg/ml in salt-free HBS p24: p24, 10 µg/mlin 10 mM Na-acetate, pH 5.0

Second MAb: Undiluted hybridoma supernatant containing secondMAb

HCl: 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 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 super-

natant, 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).

• 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.

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

Results

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.

Figure 3Reactivity pattern matrixshowing the bindingability of pairs of MAbs to p24.

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 multi-

determinant 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.

Figure 4Grouping 30 MAbsaccording to theirreactivity patternsidentifies 17 proposedepitope regions.

Figure 5Two-dimensional‘‘surface-like’’ map ofthe epitopes based onthe matrix in Figure 4.Overlapping circlesrepresent MAb groupswithin which pairs ofMAbs cannot bindsimultaneously.

Figure 6Multi-determinantbinding of MAbs top24. The MAbs injected at each stageare identified withreference to thediagram obtained fromtwo-site specificitystudies.

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.

Results

References

1. Van Regenmortel, M.H.V., Phil. Trans. R. Soc.Lond. B323; 451 (1989).

2. Krummenacher, C. et al. J Virology 74; 10863(2000)

3. Novotny, L. A. et al. Infect Immun 68; 2119(2000)

4. Goding, J.W., Monoclonal Antibodies:Principles and Practice (Academic Press, London,1983).

5. Fägerstam, L.G., Techniques in ProteinChemistry II, ed. J. J. Villafranca, pp. 65-71(Academic Press, New York 1991).

6. Jönsson U. et al., BioTechniques 11; 620(1991).

7. Kretschmann, E. and Raether, H., Z.Naturforschung, Teil. A 23; 2135 (1968).

8. Liedberg, B., Nylander, C. and Lundström, I.,Sensors and Actuators 4; 299 (1983).

9. Stenberg, E. et al., J. Colloid and InterfaceScience 143; 513 (1991).

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