Identification of Tn Antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies

Abstract

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody, and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors, and represent promising tools for Tn biomarker discovery independently of recognition of IgA1. [191 words].

SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors

“…The Western blot and lectin blot analysis were performed following the protocols published previously 46 . The proteins were under the following concentrations: DC-SIGN, L-SIGN, Dectin-2, MGL all at 1 μg/ml, MR at 2.5 μg/ml, biotinylated plant lectins GNA and VVA both at 1 μg/ml, Con A at 0.1 μg/ml, or antibodies mAb100 at 1 μg/ml and anti-spike antibody 1A9 at 0.5 μg/ml.…”

“…The ELISA assay was performed following the protocols published previously 46 . The S-protein trimer was immobilized in a 96-well plate (0.2 μg/well) overnight at 4°C.…”

“…The cell extracts either from SARS-CoV-2 infected or mock infected Vero E6 cells (both at 50 µg protein/lane), together with the culture supernatant (at 12 µg (2×) and 120 µg (20×)protein/lane), were loaded on the SDS-PAGE gel following addition of 4× laemmli loading buffer and 10 min heating. The gels were either stained with Coomassie Brilliant Blue or Colloidal Blue (both from Thermo Fisher Scientific) to visualize the proteins, or transferred to a PVDF membrane (Thermo Fisher Scientific) for Western blots.The Western blot and lectin blot analysis were performed following the protocols published previously46 . The proteins were under the following concentrations: DC-SIGN, L-SIGN, Dectin-2, MGL all at 1 µg/ml, MR at 2.5 µg/ml, biotinylated plant lectins GNA and VVA both at 1 µg/ml, Con A at 0.1 µg/ml, or antibodies mAb100 at 1 µg/ml and anti-spike antibody 1A9 at 0.5 µg/ml.…”

Cosmc controls B cell homing

“…Antibodies used in ELISA are from Southern Biotech or Thermo Scientific. Anti-Tn antibody prepared in the lab 57 , was conjugated to Alexa Fluor 647 according to manufacturer’s protocol (Thermo Scientific A20173). Biotinylated lectins were purchased from Vector Laboratories and incubated with final concentration at 2 µg per ml.…”

Towards structure-focused glycoproteomics

“…Furthermore, recombinant antibodies displaying affinity towards glycoepitopes are relevant tools for structure-focused glycoproteomics [ 152–157 ]. For example, the recombinant mouse anti-Tn IgM ReBaGs6 was used to immunoprecipitate Tn-containing glycoproteins from Tn-positive Colo205 SimpleCells lysates for downstream mass spectrometry identification [ 154 ]. Smart anti-glycan reagents (SAGRs) represent another exciting type of emerging affinity tools [ 156 , 157 ].…”