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. Author manuscript; available in PMC: 2019 Jun 24.
Published in final edited form as: Methods Mol Biol. 2019;1955:287–308. doi: 10.1007/978-1-4939-9148-8_22

Fig. 2.

Fig. 2.

Purification and immunoreactivity analysis of tGPI-mucins. (a) General scheme for purification and functional analysis of tGPI-mucins. Parasites are first delipidated, followed by extraction of GPI-APs with 9% 1-BuOH. GPI-APs are enriched by 1-BuOH/H2O partition, and tGPI-mucins are purified by hydrophobic-interaction chromatography (HIC) with Octyl-Sepharose. The tGPI-mucin-containing fractions are detected by CL-ELISA using Ch anti-α-Gal Abs. Positive fractions are combined, lyophilized, resuspended in 1 mL 20% 1-PrOH and quantified by myo-inositol analysis, as described [19]. The purified tGPI-mucins are titrated with a pool of sera from patients with chronic ChD (ChSP) and a pool of normal human sera (NHSP), before further assays with individual sera from ChD patients. (b) Chromatography elution profile of tGPI-mucins on OS-HIC. α-Gal-Enriched fractions (lyso-tGPI-mucins, tGPI-mucins, and tGIPLs) are located by CL-ELISA with Ch anti-α-Gal Abs. RLU, relative luminescence units. (c) Titration of the tGPI-mucins was performed with a pool of sera from patients with chronic ChD (ChSP) (n=10) and a pool of normal human sera (NHSP) (n=10), at different dilutions. Ch and NHS anti-α-Gal antibodies were purified from ChSP and NHSP, as described [19]. (d) Reactivity of purified tGPI-mucins with individual sera from patients with chronic ChD (ChS), ChSP, and NHSP. The tGPI-mucin CL-ELISA titer was calculated by dividing the test sample average RLU value by the cutoff, calculated as the mean value of 10 NHSP + 10 SD. Horizontal black line: titer equal to 1.0. Green dashed line: titer <1.0 and equal or greater than 0.9 [24]. Data interpretation: positive result: titer ≥ 1.0; inconclusive result: titer <1.0, >0.9; negative result: titer <0.9. ChS and ChSP samples (c, d) were from IMT/FM/UCV, and NHSP sample (d) was from ISGlobal-Barcelona. Sera were collected from patients strictly following the International Ethical Guidelines for Biomedical Research Involving Human Subjects and protocols approved by the Institutional Review Boards of IMT/FM/UCV and ISGlobal-Barcelona.