Epitope Mapping
Screening Overlapping Peptides

Introduction

Luminex Corporation has developed a multiplexed epitope mapping assay to demonstrate the utility of the FlowMetrix™ system for determining antibody specificity. A monoclonal antibody (MAB 384) with known specificity for a region of human myelin basic protein (MBP) was used as a model. Different FlowMetrix microsphere sets were coated with one of nine different overlapping octapeptides that comprise the known region of MAB 384 specificity. The MAB 384 antibody was conjugated with a green fluorescent tag and used to identify the microsphere set displaying the specific epitope of MAB 384. Figure 1 demonstrates that the FlowMetrix system is precise and capable of rapidly determining antibody specificity. In addition to monoclonal antibodies, this assay format also can be utilized to determine the specificity of bioengineered antibodies such as single chain antibodies or antibody binding domains generated through phage display.

Figure 1

Materials

Monoclonal antibody, MAB 384, was purchased from Chemicon International, Inc., Temecula, CA. Biotinylated peptides were synthesized by Dr. Richard Cook, Baylor University School of Medicine, Houston, TX. Neutravidin, EDC (1-ethyl-3-3 [3-dimethylaminopropyl] carbodiimide hydrochloride), and Sulfo-NHS (N-hydroxysulfosuccinimide) were purchased from Pierce Chemicals, Rockford, IL. Bodipyâ FL-X was purchased from Molecular Probes, Inc., Eugene, OR.

Methods

Antibody labeling: MAB 384 (reporter) was labeled with Bodipy FL-X using methods described by the manufacturer.

Avidin Conjugation to Microspheres: Nine different sets of FlowMetrix microspheres were conjugated separately to Neutravidin (deglycosylated avidin) with a two-step EDC coupling method. Twenty microliters (8.4 million microspheres) of each microsphere type was activated for twenty minutes, washed, and suspended in a 0.25 mg/ml solution of Neutravidin in PBS. After two hours, the microspheres were washed and stored in 0.02% Tween 20, 1 mg/ml BSA in PBS, pH 7.4 (PBSTB).

Peptide Attachment to Microspheres: Each of the nine Neutravidin-coated microsphere sets was treated separately with one of the nine biotinylated peptides (Table 1). Ten microliters of biotinylated peptide at 100 - 200 ng/ml was mixed with ten microliters of microspheres and reacted for 5 minutes. The peptide loaded microspheres (targets) were washed and suspended in PBSTB.

Multiplex assay: Ten microliters of each of the nine peptide loaded microspheres were mixed. Ten microliters of the mixture was reacted with ten microliters of reporter MAB 384 at 15.5 m g/ml in PBSTB for15 minutes and assayed in fifteen to twenty seconds using the FlowMetrix System.

TABLE 1

Results

Description of Peptides to be Screened: The amino acid sequence of the region of MBP was determined using the published amino acid sequence (Roth, H.J., et al., J. Neurosci.Res.. 17, 321-328, 1990). Table 1 shows the amino acid sequence of the nine overlapping peptides produced for the screening assay. Note that to the carboxy-terminal end of all peptides was added a glycine (G)-lysine (K)-biotin.

Multiplexed Epitope Mapping: Results of the assay are shown in Figure 1. Note that the peptides overlapping peptide #5 had little or no reactivity with MAB 384. The epitope of MAB 384 was located to a linear region from amino acid 67-74 (peptide#5).

Conclusions

This epitope mapping example demonstrated the useful application of FlowMetrix technology to the area of combinatorial screening. The peptide carrying the epitope for the mouse monoclonal antibody screened in this example was clearly identified in a set of nine peptides. The identification was further shown to be specific by competitive inhibition with soluble epitope peptide. In addition, the stability of the avidin-biotin interaction for use with the FlowMetrix System was demonstrated in an excess of free biotin. This assay was developed at Luminex Corporation for the purpose of demonstrating the power of FlowMetrix technology. In addition to epitope mapping, this exciting technology can be applied to immunoassays, enzyme assays and nucleic acid based genetic analyses.

Luminex Corporation provides the system including microspheres in a format ready to apply to your specific application. Using this Assay Development Kit, your research group can develop multiplexed assays of interest in a matter of days. For further information in the US call toll free 1-888-219-8020, outside the US call 1-512-219-8020 or email scombs@luminexcorp.com.

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