Characterization of a 35-kDa carbohydrate larval antigen (CarLA) from Trichostrongylus colubriformis; a potential...

In an accompanying paper we show that antibodies in intestinal mucus that recognize a 35-kDa antigen from the surface of the L3 stage of the sheep intestinal nematode parasite, Trichostrongylus colubriformis, are strongly associated with immune rejection of L3 in a truncated infection model of immunity in sheep. Monoclonal antibody PAB-1 was used to immunopurify this antigen from T. colubriformis L3. The antigen is resistant to digestion with a range of proteases including proteinase K and does not stain on gels or blots treated with protein-detecting reagents but does stain with carbohydrate-detecting reagents. The antigen is also resistant to degradation by the action of lipases and is not soluble in organic solvents, suggesting that lipid components are not present or not accessible. Treatment with glycosidases does not affect the antigen, indicating either that sialic acid and N-linked or O-linked sugars are not present or that they are not accessible to enzyme attack. The antigen is not destroyed by harsh alkaline degradation with up to 8 m NaOH with or without borohydride reducing agent, or by extensive hydrazinolysis. Strong acid hydrolysis with trifluoroacetic acid or boiling in hydrochloric acid for 20 min does destroy the antigen. The antigen migrates as a poorly defined high molecular weight complex on native electrophoresis gels, but is detected as a major band at 35 kDa on SDS PAGE either by carbohydrate staining or by immunoblotting with antibody from immune sheep intestinal mucus and with mAb PAB-1. Proteinase K digestion and alkaline degradation of extracts from L3 of 10 other parasitic nematode species revealed that L3 of each species contained a carbohydrate staining molecule which can be detected by mAb PAB-1 and by mucus antibody from immune sheep. Because antibodies in intestinal mucus are directed against these antigens and may be responsible for protective immunity, carbohydrate larval antigens (CarLA) could represent a new family of molecules with potential as targets for stimulating host immunity.