Session: Session 1
Parasitic Disease Diagnosis: Iterative IgG Separation for Enhanced Accuracy
The IgName ANR project aims to identify T. gondii (Tg) differentially specific IgG from a mother and her child in a newborn's serum as a new tool for neonatal serological diagnosis of congenital toxoplasmosis.
The diagnosis relies on detecting specific antibodies (Ab) against Tg newly synthesized by the newborn. Since maternal Ab are still present in newborn blood during the first few months of life, detecting Ab against Tg is insufficient to evidence a congenital transmission of toxoplasmosis to the child. However, a distinction between maternal and child Ab can be made by mass spectrometry (MS) since they differ by their sequence.
Igs are glycoproteins from body fluids, comprising four polypeptide chains (two light, two heavy). Among them, IgG classes consist of four subclasses encoded by the IGHG gene, resulting in amino acid polymorphism region within the Fc region of heavy chains. Characterizing serum IgG's Fc region would allow the differentiation of alleles of IgG subclasses and, consequently differentiation of maternal from newborn IgG. Identifying Tg-specific IgGs requires serum purification and prefractionation before nanoLC-MS analysis to avoid co-elution of different human Fc/2.
Following our strategy, parasitic lysate extract was filtered on a Ni-NTA column grafted with Ab directed against Tg proteins, then fractionated using nanobodies (CaptureSelect, Affinity Matrix) for each IgG to enrich Fc/2. IGHG allele detection after purification is ensured by middle-down analysis after IdeS treatment on an HESI-Orbitrap Tribrid Eclipse mass spectrometer (ThermoFisher) with UHPLC separation on a Poroshell 300SB-C3 column (Agilent).
Our protocol, downscaled for tiny serum quantities, is compatible with newborn samples (few µL). The iterative purification was first validated by bottom-up proteomics, revealing several alleles from the mixture. The study further focused on middle-down analysis to identify subclasses and validate specific Tg antibody extraction.
The diagnosis relies on detecting specific antibodies (Ab) against Tg newly synthesized by the newborn. Since maternal Ab are still present in newborn blood during the first few months of life, detecting Ab against Tg is insufficient to evidence a congenital transmission of toxoplasmosis to the child. However, a distinction between maternal and child Ab can be made by mass spectrometry (MS) since they differ by their sequence.
Igs are glycoproteins from body fluids, comprising four polypeptide chains (two light, two heavy). Among them, IgG classes consist of four subclasses encoded by the IGHG gene, resulting in amino acid polymorphism region within the Fc region of heavy chains. Characterizing serum IgG's Fc region would allow the differentiation of alleles of IgG subclasses and, consequently differentiation of maternal from newborn IgG. Identifying Tg-specific IgGs requires serum purification and prefractionation before nanoLC-MS analysis to avoid co-elution of different human Fc/2.
Following our strategy, parasitic lysate extract was filtered on a Ni-NTA column grafted with Ab directed against Tg proteins, then fractionated using nanobodies (CaptureSelect, Affinity Matrix) for each IgG to enrich Fc/2. IGHG allele detection after purification is ensured by middle-down analysis after IdeS treatment on an HESI-Orbitrap Tribrid Eclipse mass spectrometer (ThermoFisher) with UHPLC separation on a Poroshell 300SB-C3 column (Agilent).
Our protocol, downscaled for tiny serum quantities, is compatible with newborn samples (few µL). The iterative purification was first validated by bottom-up proteomics, revealing several alleles from the mixture. The study further focused on middle-down analysis to identify subclasses and validate specific Tg antibody extraction.