ABSTRACT
BACKGROUND: Rheumatoid factor (RF) consists of autoantibodies that bind the fragment crystallizable (Fc) region of human immunoglobulin G (IgG) and present in sera of rheumatoid arthritis (RA) patients. Immunoassays to detect antidrug antibodies (ADA) in RA patient samples may experience interference due to RF binding and crosslinking Fc regions of the capture and detection antibody reagents. To overcome this interference, a novel Fab affinity-capture and elution (ACE)-bridging immunoassay (Fab ACE-Bridge) was developed with monovalent-recombinant Fab to avoid RF interference. METHODS: ACE and ACE-Bridge assays were developed to detect ADA against a therapeutic monoclonal antibody using samples from healthy donors, psoriasis patients, and RA patients. The performance of these assays was compared to a novel Fab ACE-Bridge assay, in which monoclonal antibody was replaced with monovalent Fab. RESULTS: High screening signals in the ACE and ACE-Bridge assays were detected in RA patient samples but not in samples from healthy donors or psoriasis patients. The high screening signals in RA samples did not inhibit to the expected extent in the confirmatory assay, a consistent feature of false-positive screening results. Further investigation revealed RF as the interferent affecting assay performance. Modification of the ACE-Bridge assay by using monovalent Fab eliminated RF interference while allowing for sensitive and drug-tolerant detection of authentic ADA. CONCLUSIONS: RF interfered significantly in traditional ACE and ACE-Bridge assays. Implementation of a novel monovalent Fab ACE-Bridge assay overcame RF interference. The use of monovalent Fab is recommended for immunogenicity assays when assessing ADA in RA patient samples.
Subject(s)
Arthritis, Rheumatoid , Rheumatoid Factor , Humans , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/drug therapy , Immunoassay/methods , Immunoglobulin G , Antibodies, MonoclonalABSTRACT
Enteroendocrine cells (EECs) are a minor cell population in the intestine yet they play a major role in digestion, satiety and nutrient homeostasis. Recently developed human intestinal organoid models include EECs, but their rarity makes it difficult to study their formation and function. Here, we used the EEC-inducing property of the transcription factor NEUROG3 in human pluripotent stem cell-derived human intestinal organoids and colonic organoids to promote EEC development in vitro An 8-h pulse of NEUROG3 expression induced expression of known target transcription factors and after 7â days organoids contained up to 25% EECs in the epithelium. EECs expressed a broad array of human hormones at the mRNA and/or protein level, including motilin, somatostatin, neurotensin, secretin, substance P, serotonin, vasoactive intestinal peptide, oxyntomodulin, GLP-1 and INSL5. EECs secreted several hormones including gastric inhibitory polypeptide (GIP), ghrelin, GLP-1 and oxyntomodulin. Injection of glucose into the lumen of organoids caused an increase in both GIP secretion and K-cell number. Lastly, we observed formation of all known small intestinal EEC subtypes following transplantation and growth of human intestinal organoids in mice.
Subject(s)
Enteroendocrine Cells/cytology , Enteroendocrine Cells/metabolism , Pluripotent Stem Cells/cytology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Count , Cell Differentiation , Hormones/metabolism , Humans , Intestines/cytology , Nerve Tissue Proteins/metabolism , Organoids/cytology , Pluripotent Stem Cells/metabolism , Time Factors , Transcription Factors/metabolismABSTRACT
Activation of free fatty acid receptor 1 (GPR40) by synthetic partial and full agonists occur via distinct allosteric sites. A crystal structure of GPR40-TAK-875 complex revealed the allosteric site for the partial agonist. Here we report the 2.76-Å crystal structure of human GPR40 in complex with a synthetic full agonist, compound 1, bound to the second allosteric site. Unlike TAK-875, which acts as a Gαq-coupled partial agonist, compound 1 is a dual Gαq and Gαs-coupled full agonist. compound 1 binds in the lipid-rich region of the receptor near intracellular loop 2 (ICL2), in which the stabilization of ICL2 by the ligand is likely the primary mechanism for the enhanced G protein activities. The endogenous free fatty acid (FFA), γ-linolenic acid, can be computationally modeled in this site. Both γ-linolenic acid and compound 1 exhibit positive cooperativity with TAK-875, suggesting that this site could also serve as a FFA binding site.
