Comparative analysis of two meningococcal immunotyping monoclonal antibodies by resonant mirror biosensor and antibody gene sequencing

Abstract

Lipooligosaccharide (LOS) is a major surface component of the cell walls of Neisseria meningitidis, which is important for its roles in pathogenesis and antigenic variation, as a target for immunological typing, and as a possible vaccine component. Although the structures of many antigenic variants have been determined, routine immunological typing of these molecules remains problematic. Resonant mirror analysis was combined with gene sequencing to characterize two monoclonal antibodies (MAbs) used in typing panels that were raised against the same LOS immunotype, L3,7,9. The two MAbs (MAb 4A8-B2 and MAb 9-2-L379) were of the same immunoglobulin subtype, but while MAb 9-2-L379 was more than a 1,000-fold more sensitive in immunotyping assays of both whole meningococcal cells and purified LOS, MAb 4A8-B2 was more specific for immunotype L3,7,9. The differences in sensitivity were a consequence of MAb 9-2-L379 having a 44-fold-faster association constant than MAb 4A8-B2. Comparison of the amino acid sequences of the variable chains of the MAbs revealed that they had very similar heavy chains (81% amino acid sequence identity) but diverse light chains (54% sequence identity). The differential binding kinetics and specificities observed with these MAbs were probably due to differences in the epitopes recognized, and these were probably a consequence of the different immunization protocols used in their production.

FIG. 1

ELISA of MAbs 4A8-B2 and 9-2-L379 against N. meningitidis cells and purified L3,7,9 LOS. The relative binding of MAb 9-2-L379 to purified LOS (○) and whole cells (●) and MAb 4A8-B2 to purified LOS (□) and whole cells (■) of the same meningococcal isolate are shown. Error bars represent the standard deviation of triplicate determinations.

FIG. 2

Real-time interaction curves for MAbs 4A8-B2 and 9-2-L379 against immobilized L3,7,9. Real-time binding interaction curves for various concentrations of each MAb with purified LOS of immunotype L3,7,9 are shown. (A) Overlay of the binding curves of MAb 4A8-B2 at 0.07, 0.17, 0.35, 0.52, 0.70, and 1.05 μM. (B) Overlay of the binding curves of the MAb 9-2-L379 at 0.005, 0.01, 0.02, 0.03, 0.10, and 0.25 μM. For clarity, not all of the binding data collected are shown, and the dissociation curves have been omitted.

FIG. 3

Binding kinetics for MAbs 4A8-B2 and 9-2-L379 against immobilized L3,7,9 LOS. The association rates (K_ON) of MAb 4A8-B2 (●) and MAb 9-2-L379 (○) with purified LOS at 25°C, plotted against MAb concentration, are shown. The K_ass constant of each MAb was derived from the gradient of the slope.

FIG. 4

Primary sequence comparison of the variable regions within MAbs 4A8-B2 and 9-2-L379. The aligned, deduced primary sequences of the variable regions from MAbs 4A8-B2 and 9-2-L379 are shown. The amino acid sequences corresponding to MAb 9-2-L379 are compared with those of MAb 4A8-B2. Amino acid identities are represented with a period, and differences are indicated by the appropriate letter; hyphens represent deletions relative to the sequence of MAb 4A8-B2. The sequences are numbered according to the Kabat database (9), with lowercase letters representing variable-length insertion sequences. CDRs are indicated by white text on a black background.

FIG. 5

Sugar structures of LOS molecules known to interact with MAbs 9-2-L379 and 4A8-B2. The structures of LOS molecules associated with the immunotypes L2, L3,7,9, L5, and L8 are given (7, 15, 18, 22), with the sugar moieties that are likely to be involved in the binding interactions that distinguish the two MAbs highlighted. Those moieties putatively contributing to the epitope recognized by MAb 9-2-L379 are shown with boldface text, while those that might play a role in the more specific interaction of MAb 4A8-B2 with L3,7,9 LOS are shown boxed. The PEA group that is potentially involved in binding of both antibodies to LOS immunotype L3,7,9 is shown boxed in boldface text.