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Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

Maass DR, Harrison GB, Grant WN, Hein WR, Shoemaker CB - PLoS Pathog. (2009)

Bottom Line: Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms.Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations.These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Science and Research Ltd, Porirua, Wellington, New Zealand.

ABSTRACT
The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

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Blocking ELISA for epitope analysis of the anti-T. colubriformis CarLA scFvs.Phage displaying scFv (top row) was added to wells coated with T. colubriformis extract that had been pre-treated with soluble expressed scFv (first column). Blocking by the soluble scFvs is reflected by substantially reduced signals produced by the phage-displayed scFvs. Values represent percent binding compared to PBS controls. Wells displaying greater than 70% signal reduction (blocking) are boxed. The assay was performed in duplicate on at least three separate occasions.
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ppat-1000597-g002: Blocking ELISA for epitope analysis of the anti-T. colubriformis CarLA scFvs.Phage displaying scFv (top row) was added to wells coated with T. colubriformis extract that had been pre-treated with soluble expressed scFv (first column). Blocking by the soluble scFvs is reflected by substantially reduced signals produced by the phage-displayed scFvs. Values represent percent binding compared to PBS controls. Wells displaying greater than 70% signal reduction (blocking) are boxed. The assay was performed in duplicate on at least three separate occasions.

Mentions: Blocking and additive ELISA methods were employed to establish epitope grouping among the anti-CarLA scFvs. Figure 2 shows the results of blocking ELISAs in which each of the nine purified recombinant anti-CarLA scFvs, or a control anti-sheath scFv, Tc.2 [12], were tested for the ability to block the binding of the phage displayed scFvs to extracts containing CarLA. This analysis identified three distinct epitope groups. Group E1 was comprised of Tc.A6, Tc.B2 Tc.C1and Tc.C10; group E2 included Tc.C2 Tc.D1 and Tc.E9; and group E3 contained Tc.C3 and Tc.E6. As expected, Tc.2 phage binding, which binds to a sheath antigen that is also present in the extracts, was not inhibited by any of the soluble scFvs. These results were independently confirmed using additive ELISA methods [14] (Figure S1). Two representatives of scFvs from group E1, E2 and E3 were demonstrated by ELISA to recognize CarLA that was purified by immunoaffinity with the anti-CarLA mAb, PAB-1 [9]. The scFvs, Tc.1 and Tc.2 which recognize other surface antigens in crude extracts [12], did not react with the PAB1 purified CarLA thus confirming that the target of the nine scFvs is CarLA (Table S1).


Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

Maass DR, Harrison GB, Grant WN, Hein WR, Shoemaker CB - PLoS Pathog. (2009)

Blocking ELISA for epitope analysis of the anti-T. colubriformis CarLA scFvs.Phage displaying scFv (top row) was added to wells coated with T. colubriformis extract that had been pre-treated with soluble expressed scFv (first column). Blocking by the soluble scFvs is reflected by substantially reduced signals produced by the phage-displayed scFvs. Values represent percent binding compared to PBS controls. Wells displaying greater than 70% signal reduction (blocking) are boxed. The assay was performed in duplicate on at least three separate occasions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742895&req=5

ppat-1000597-g002: Blocking ELISA for epitope analysis of the anti-T. colubriformis CarLA scFvs.Phage displaying scFv (top row) was added to wells coated with T. colubriformis extract that had been pre-treated with soluble expressed scFv (first column). Blocking by the soluble scFvs is reflected by substantially reduced signals produced by the phage-displayed scFvs. Values represent percent binding compared to PBS controls. Wells displaying greater than 70% signal reduction (blocking) are boxed. The assay was performed in duplicate on at least three separate occasions.
Mentions: Blocking and additive ELISA methods were employed to establish epitope grouping among the anti-CarLA scFvs. Figure 2 shows the results of blocking ELISAs in which each of the nine purified recombinant anti-CarLA scFvs, or a control anti-sheath scFv, Tc.2 [12], were tested for the ability to block the binding of the phage displayed scFvs to extracts containing CarLA. This analysis identified three distinct epitope groups. Group E1 was comprised of Tc.A6, Tc.B2 Tc.C1and Tc.C10; group E2 included Tc.C2 Tc.D1 and Tc.E9; and group E3 contained Tc.C3 and Tc.E6. As expected, Tc.2 phage binding, which binds to a sheath antigen that is also present in the extracts, was not inhibited by any of the soluble scFvs. These results were independently confirmed using additive ELISA methods [14] (Figure S1). Two representatives of scFvs from group E1, E2 and E3 were demonstrated by ELISA to recognize CarLA that was purified by immunoaffinity with the anti-CarLA mAb, PAB-1 [9]. The scFvs, Tc.1 and Tc.2 which recognize other surface antigens in crude extracts [12], did not react with the PAB1 purified CarLA thus confirming that the target of the nine scFvs is CarLA (Table S1).

Bottom Line: Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms.Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations.These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Science and Research Ltd, Porirua, Wellington, New Zealand.

ABSTRACT
The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

Show MeSH
Related in: MedlinePlus