Optimisation of lectin binding conditions

Initial experiments tested the conditions for lectin binding to sheathed T. circumcincta and H. contortus larvae. Binding of five lectins (DBA; SBA, BGS II, WGA and ConA) to sheathed larvae was tested in PBS as lectin binding buffer, as described for other nematode species (KUMAR and PRITCHARD 1992; NAYAR et al. 1995) and life cycle stages (PALMER and MCCOMBE 1996). The binding of lectins to L3 in this medium was inconsistent, as not all larvae were labelled and the fluorescence resulting from the binding was very weak (Fig. 4, 5) and could only be observed under high magnification. Binding results were not improved by exsheathing larvae (Tab. 10). In contrast, using the same protocol lectins bound much more intensely to

the surface of adult worms (Fig. 6), showing the assay conditions were adequate for this stage of development. Binding to adult worms was across the entire surface of the worm, revealing a striated pattern (Fig. 6). Since the lectin binding did not occur to all larvae examined (referred to as inconsistent or low consistency of binding hereafter) and pattern and intensity on the larvae were not ideal for identification and differentiation of species, several modifications to assay conditions were tested in an attempt to improve the lectin binding to larvae, using adult worms as positive control.

The lectin binding inhibition tested by pre-incubation of the lectins with their specific monosaccharide prior to the binding experiments on H. contortus adults showed that most lectins were stopped from binding, as no fluorescence on the surface was observed in PBS as well as HEPES buffer (Tab. 15). This indicated the high specificity to the corresponding carbohydrate moiety of the lectins. Only PNA (GalNAc) and WGA (GlcNAc) were not inhibited by addition of the monosaccharides as the adults still exhibited very weak (+) and weak (++) fluorescence with those lectins (Tab. 15), but it was highly reduced in comparison with binding without previous inhibition of bright fluorescence (++++) (Tab. 22). It was thought that plant parasitic nematodes may bind lectins more effectively than animal parasitic nematodes based on the results of MCCLURE and STYNES (1988). That was not observed in the present study (Tab. 24).

Lectin binding in different buffers and with the addition of cations was investigated.

The recommended cations (Tab. 9) were added to PBS, triton buffer and HEPES in a final concentration of 0.1 mM Ca²⁺ and 0.01 mM Mn²⁺. Triton buffer, although recommended by Sigma Chemical Company for the use as lectin buffer, caused visible precipitation with Ca²⁺ and Mn²⁺ at the concentration and pH specified and was therefore not used further. HEPES buffer is recommended by the lectin suppliers, Vector labs, as it should not precipitate Ca²⁺ and Mn²⁺. Generally, the addition of cations to PBS buffer made little difference to lectin binding to T.

circumcincta adults (Tab. 14). In fact, DBA, PHA E+L, LTL and UEA showed reduced binding after the addition of ions to PBS. Overall, addition of ions to HEPES improved binding only for SBA, but generally did not decrease binding, as observed with PBS

plus ions, only SNA binding was slightly reduced (Tab. 14). For lectins which did not require the addition of ions, binding results were similar in HEPES and PBS or slightly better in HEPES, especially when tested on sheathed T. circumcincta larvae (Tab.11, 12) and HEPES was therefore established as the standard buffer for all lectin binding except for PTL II where PBS was preferred (Tab. 12). Similar results for lectins with additional ions were also observed in sheathed T. circumcincta larvae (Tab. 13) where a higher percentage of larvae showed binding to the surface with some lectins (SBA, WGA, ConA) when ions were added to the buffer solution, but the binding was still variable and inconsistent (Tab. 13) Although the addition of ions caused little improvement in lectin binding to adults and to sheathed T. circumcincta larvae it was continued according to the manufacturer’s recommendation, to ensure the ion requirements of the lectins were met.

To remove any possible dirt or detritus from the surface of L3, especially lipid soluble structures which may have obscured binding to carbohydrate groups on the surface of the sheath, various washing techniques were employed. These included increased washing with PBS and use of mild and strong detergents. None of these treatments increased lectin binding to L3. Triton X-100 reduced lectin binding to the surface of adult worms, suggesting that strong detergents could remove carbohydrate groups from the surface. Larvae were also washed, but without positive effects. This is consistent with BLAXTER et al. (1992) who reported glycoproteins were mostly present on the surface coat of nematodes which could be washed off with harsh detergents.

Attempts were made to reduce possible effects of shedding of bound lectins by sheathed L3. Antibodies have been shown to be actively shed from the surface of both the free living nematode C. elegans (POLITZ and PHILLIP 1992) and T. canis larvae (SMITH et al. 1981), while increased antibody binding to the surface of immobilised T. canis L2 has been observed (SMITH et al. 1981). The shedding of antibodies was very rapid, occurring within 3 hours from T. canis larvae (SMITH et al.

1981). This suggests that shedding from the cuticle is an active process and requires

metabolic activity. This is in agreement with SMITH et al. (1981) and MAIZELS et al.

(1984) who showed that metabolic inhibitors and cooling reduced antibody shedding by T. canis. Similarly, the monoclonal antibody 8D bound well to the surface coat of H. contortus, T. spiralis and M. incognita when they were processed in cryosections, but was not detected on live nematodes and the shedding from the surface coat after incubation with the antibody 8D was observed in M. incognita (DE MENDOZA 1999).

As the antigens being shed have been reported to be heavily glycosylated (MAIZELS and PAGE, 1990), they might be the target molecules bound by lectins. If so, and these glycosylated antigens are loosely attached to the surface and are frequently shed, this may explain the patchy and inconsistent binding of lectins to the surface of sheathed L3 observed in this study (Tab. 16, Tab. 17, Fig. 4, Fig. 8, Fig. 9). Also the imprint of the adult worms on the cover slip (Fig. 24) may be an indication, that the carbohydrates are only loosely attached and only on the surface of the adult worms.

When the imprint occurred, no fluorescence was retained in that surface area of the worm (Fig. 24). L3 were immobilised and killed with liquid nitrogen (3.1.11), which increased the consistency of lectin binding to the amphids, implying that immobilisation had a positive effect on the lectin binding observed.