Synthetic nanobodies as tools to distinguish IgG Fc glycoforms.

Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.

Combining urine lipoarabinomannan with antibody detection as a simple non-sputum-based screening method for HIV-associated tuberculosis.

BACKGROUND: Simple methods for the accurate triaging and screening of HIV-associated tuberculosis (TB) are urgently needed. We hypothesized that combining serum antibody with urine lipoarabinomannan (U-LAM) detection can improve the detection of HIV-associated TB. METHODS: We performed a case-control study with sampling from a prospective study of South African HIV-infected subjects who were screened for TB prior to initiating antiretroviral therapy. Sera from all available TB cases (n = 74) and randomly selected non-TB controls (n = 30), all tested for U-LAM, sputum microscopy, GeneXpert, and cultures, were evaluated for antibodies to LAM and arabinomannan (AM). Diagnostic logistic regression models for TB were developed based on the primary test results and the additive effect of antibodies with leave-one-out cross-validation. RESULTS: Antibody responses to LAM and AM correlated strongly (p<0.0001), and IgG and IgM reactivities were significantly higher in TB than non-TB patients (p<0.0001). At 80% specificity, the target specificity for a non-sputum-based simple triage/screening test determined by major TB stakeholders, combining U-LAM with IgG detection significantly increased the sensitivity for HIV-associated TB to 92% compared to 30% for U-LAM alone (p<0.001). Sputum microscopy combined with IgG detection increased sensitivity to 88% compared to 31% for microscopy alone, and Xpert with IgG increased sensitivity to 96% and 99% compared to 57% for testing one, and 70% for testing two sputa with Xpert alone, respectively. CONCLUSION: Combining U-LAM with serum antibody detection could provide a simple low-cost method that meets the requirements for a non-sputum-based test for the screening of HIV-associated TB.