Fig 2: Immunocapture of drug/TNF complexes by Type 3 antibodies. Type 3 anti-golimumab/TNF antibody AbD25705 and Type 3 anti-adalimumab/TNF antibody AbD20350 were coated on a microtiter plate at 1 µg/mL. Golimumab and adalimumab at a fixed concentration (1 µg/mL = 7 nM) were incubated for 1 hour with an increasing amount of TNF and added to the plate. Detection was performed with anti-human IgG:HRP antibody specific for the CH2 domain that detects golimumab or adalimumab but not the Fab antibodies, followed by QuantaBlu fluorogenic peroxidase substrate.

Fig 3: Comparison of assay formats. (a) Schematic view of the assay set-up: bridging assay set-up using a Type 1 reagent for capture and an HRP-conjugated Type 1 antibody for detection of a bivalent drug and (b) antigen capture assay using an HRP-conjugated Type 3 antibody for detecting a monovalent or bivalent drug in complex with its drug target. (c) Detection of adalimumab using a Type 3 antibody in antigen capture assay (black), or Type 1 capture and detection antibodies in a bridging assay (red). For the antigen capture assay, human TNF was coated at 5 µg/mL on a microtiter plate overnight. After washing and blocking, adalimumab spiked into 10% human serum was added. HRP-conjugated anti-adalimumab/TNF antibody AbD18754 (KD = 67 nM) was added at 2 µg/mL in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate. For the adalimumab bridging assay, the anti-adalimumab Type 1 antibody AbD18654 (KD = 0.16 nM) was coated at 1 µg/mL on a microtiter plate overnight. After washing and blocking, adalimumab spiked into 10% human serum was added, followed by HRP-conjugated Type 1 anti-adalimumab antibody AbD18655 (KD = 0.06 nM) at 2 µg/mL in HISPEC assay diluent and QuantaBlu fluorogenic peroxidase substrate.

Fig 4: Synergistic toxicity of iron and inflammation is mediated by maternal TNFα.a Iron-loaded hepcidin KO dams were treated intravenously via the retroorbital sinus with 500 μg neutralizing TNFα antibody (nTNFα, white circles) or isotype control IgG (IgG, blue circles) neutralizing trinitrophenol 15 h prior to subcutaneous injection of LPS (0.5 μg/g) on E15.5 for 24 h. b Gross morphology and incidence of resorbing embryos. c–e Maternal liver expression of inflammatory markers Saa1, Il6, and Cxcl9 normalized to Hprt. f–i Placental mRNA expression of inflammatory markers Tnf, Il6, Il1b, and Cxcl9 normalized to Rpl4. j, k Western blots and quantitation of whole placenta or embryo liver lysates for cleaved caspase-3 expression normalized to β-actin. l TUNEL stain of placental sections. m Immunohistochemistry for cleaved caspase-3 (brown) and CD31 (red) in paraffin-embedded placenta, embryo heart, lung, and liver. l, m Representative images of n = 3 sections/group. Scale bar = 50 μm. f–m Embryo and placentas were randomly selected for analysis. c–k Error bars represent mean ± s.e.m. Statistical differences between groups were determined by two-tailed Mann–Whitney U or two-tailed Student’s t-test (denoted by *). P-values are indicated in each figure panel. Source data are provided as a Source data file.

Fig 5: TNF-NF-κB pathway is enriched with aging in ICOS+CD38+ cTfh(A) Schematic for RNA-seq analyses.(B and C) Weighted gene correlation network analysis on ICOS+CD38+ cTfh at day 7 from young (B) and elderly (C) adults.(D) Module preservation analysis was performed using Fisher’s exact test. Heatmap color and value indicate the −log10(p value) for the overlap in genes in modules.(E) Genes ranked based on module membership for elderly module EM4. Then, GSEA was performed using the MSigDB HALLMARK collection. Positive enrichment scores indicate enrichment for genes in EM4.(F) Transcription factors with module membership >0.80 in EM4 displayed as a multiple association network (GeneMANIA without gene prediction).(G) Ingenuity Pathway Analysis for predicted upstream regulators for ICOS+CD38+ cTfh at day 7 for young and elderly adults.(H) Differentially expressed NF-κB target genes comparing ICOS+CD38+ cTfh at day 7 from elderly (purple) and young (orange) adults.(I) GSEA for NF-κB target genes to compare ICOS+CD38+ cTfh from elderly and young adults.(J) Example plot from 1 young adult and 1 elderly adult for total NF-κB p50 protein from an independent cohort of young and elderly adults. Geometric mean fluorescence intensity (MFI) shown.(K) Total NF-κB p50 for ICOS+CD38+ cTfh at baseline in young (orange) and elderly (purple) adults (n = 16 in each group), as taken from an independent cohort of young and elderly adults.(L and M) Gene expression for TNFRSF1A (L) (n = 6 for young; n = 8 for elderly) and TNFRSF1B (M) (n = 8 for elderly) shown at day 7 after vaccine from log2-transformed counts data for young (orange) and elderly (purple) adults.