One non-typable (A950002) and two THi strains (Rd and Eagan) were used in receptor overlay experiments as above (Fig. 4) . Alanine substitutions at Ile-91 confirmed previous results (Virji et al., 2000) . Taken individually, substitutions of Ile-91 for Ala or Thr abrogated binding of Fig. 3 . Relative binding of CEACAM1-Fc constructs to N. meningitidis isolates expressing distinct Opa proteins. Bacterial lysates were dotted on to nitrocellulose and overlaid with NA1B-Fc constructs as indicated. Binding relative to the native receptor was determined by densitometric analysis of immno-blots using NIH Scion Image programme. One hundred per cent binding level is indicated by the horizontal line allowing comparison to native receptor binding. Mean values and SE of > 3 replicates are shown in each case, except Q89A (n = 1) for OpaA, B and X. However, alanine substitution at all three positions confirm previous observations (Virji et al., 1999) . Black inserts in C show the levels of binding of the various receptor constructs to Opaisolate of strain C751; experiments were conducted simultaneously with C751OpaD in the presence of the receptors shown. all three strains. However, substitution I91L reduced binding only of the THi strain Eagan, suggesting a requirement for the extended hydrophobic arm of Ile in Eagan binding. Alanine substitution of Tyr-34 abrogated the binding of Rd and Eagan, whereas no effect on NTHi A950002 was observed. In contrast, Y34F substitution led to an increased binding of all three strains varying from 45 to 80% above native receptor binding levels, whereas Y34S had a differential effect on the three strains tested, ranging from no binding of Rd to a slight increase in the binding of A950002. These data broadly reflect results with neisserial Opa binding. Finally substitutions at Gln-89 show that reduction or abrogation of the side chain (Q89N and Q89A) has no deleterious effect, rather its effect is often that of enhanced binding (Fig. 4) .