Journal: Scientific Reports

Article Title: Anti-IgD nanobodies as novel tools for studying human IgD biology

doi: 10.1038/s41598-025-09118-4

Figure Lengend Snippet: Interaction of anti-IgD Nbs with IgD. Binding of anti-IgD Nbs ( a ) aδNb107, ( b ) aδNb367, ( c ) aδNb408 and ( d ) aδNb571 to IgD captured on an anti-His chip by SPR. A two-fold dilution series of TEV-cleaved anti-IgD Nbs was flowed over His-tagged IgD, from the highest concentration 200 nM (black line) to the lowest concentration 1.6 nM (purple line). Binding of IgD-Fc to anti-IgD Nbs ( e ) aδNb107, ( f ) aδNb367, ( g ) aδNb408 and ( h ) aδNb571. The His-tagged Nbs were captured by anti-His. A two-fold dilution series of IgD-Fc was flowed over, with the highest concentration 200 nM (black line) and the lowest concentration 3 nM (green line). Double-reference subtraction was not performed for ( e ) aδNb107, ( f ) aδNb367 and ( g ) aδNb408, with the 0 nM concentration shown as a gray line. RU, resonance units.

Article Snippet: Anti-IgD Nbs aδNb107 (Addgene 220319), aδNb367 (Addgene 220320), aδNb408 (Addgene 220321) and aδNb571 (Addgene 220322) in pET-15b were codon-optimized for E. coli expression and synthetized by GenScript, with an N-terminal periplasmic leader and a C-terminal TEV cleavage site, followed by a glycine-serine linker and His 6 -tag.

Techniques: Binding Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Anti-IgD nanobodies as novel tools for studying human IgD biology

doi: 10.1038/s41598-025-09118-4

Figure Lengend Snippet: aδNb408 can be used as a purification tool. ( a ) Testing of elution conditions for the aδNb408 affinity resin. Alexa Fluor 488 (AF488)-labeled IgD-Fc was detected by measuring fluorescence of combined elution fractions (left panel) or of the aδNb408 affinity resin after elution (right panel). A.U., arbitrary units. ( b ) Size exclusion chromatogram showing IgD purification from BSA-containing buffer, with elution at pH 3.5 or pH 2.5. Input represents the maximal amount of IgD that could have been purified. The elution position of monomeric BSA is indicated. Note the buffer mismatch occurring from ~ 25 min onwards. ( c ) Size exclusion chromatogram showing purification of IgD supplemented into culture supernatant containing 10% FBS at 100 nM or 200 nM, with elution at pH 3.5. Input represents the maximal amount of IgD that could have been purified. Note the buffer mismatch occurring from ~ 25 min onwards.

Article Snippet: Anti-IgD Nbs aδNb107 (Addgene 220319), aδNb367 (Addgene 220320), aδNb408 (Addgene 220321) and aδNb571 (Addgene 220322) in pET-15b were codon-optimized for E. coli expression and synthetized by GenScript, with an N-terminal periplasmic leader and a C-terminal TEV cleavage site, followed by a glycine-serine linker and His 6 -tag.

Techniques: Purification, Labeling, Fluorescence

Journal: Scientific Reports

Article Title: Anti-IgD nanobodies as novel tools for studying human IgD biology

doi: 10.1038/s41598-025-09118-4

Figure Lengend Snippet: Anti-IgD Nbs as tools for SPR capture. Biotinylated aδNb107, aδNb367 and aδNb408 were immobilized onto an SA chip. ( a ) IgD-Fc was captured by anti-IgD Nbs and a two-fold dilution series of aδNb571 was flowed over, with the highest concentration 50 nM aδNb571 (orange line) and the lowest concentration 3 nM aδNb571 (green line). ( b ) Full-length IgD, HAPPID1, was captured by anti-IgD Nbs and a two-fold dilution series of the polcalcin allergen Ole e 3 was flowed over, with the highest concentration 100 nM (cyan line) and the lowest concentration 1.6 nM (purple line). RU, resonance units.

Article Snippet: Anti-IgD Nbs aδNb107 (Addgene 220319), aδNb367 (Addgene 220320), aδNb408 (Addgene 220321) and aδNb571 (Addgene 220322) in pET-15b were codon-optimized for E. coli expression and synthetized by GenScript, with an N-terminal periplasmic leader and a C-terminal TEV cleavage site, followed by a glycine-serine linker and His 6 -tag.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Anti-IgD nanobodies as novel tools for studying human IgD biology

doi: 10.1038/s41598-025-09118-4

Figure Lengend Snippet: Anti-IgD Nb pairs as a tool for cell-binding. ( a ) Left: Flow cytometry data of AF488-labeled anti-IgD Nbs binding to Namalwa cells. A non-IgD binding Nb was used as a control for non-specific background binding. Right: MFI was normalized to the control and the highest binder aδNb367, with the mean plotted as a gray bar and the individual data points as black crosses (n = 3). ( b ) Left: Flow cytometry data of anti-IgD Nb pairs binding to Namalwa cells. A pair of non-IgD binding Nbs was used as a control for non-specific background binding. Right: MFI was normalized to the control and the highest binder aδNb408-aδNb107, with the mean plotted as a gray bar and the individual data points as black crosses (n = 2). ( c ) Comparison of the bivalent/bispecific aδNb408-Nb constructs (panel b, left) with their monovalent Nb components (panel a, left); these data are from the same experiment and are therefore comparable. MFI values of monovalent aδNb107 (orange, 662), monovalent aδNb367 (blue, 1452), monovalent aδNb408 (magenta, 1339) and monovalent aδNb571 (green, 921) are shown additively as filled bars, separated by a dashed line. MFI values of equivalent bivalent/bispecific Nbs are shown as empty black bars: aδNb408-aδNb107 (3533), aδNb408-aδNb367 (2641), aδNb408-aδNb408 (1998) and aδNb408-aδNb571 (2488). ( d ) Flow cytometry data of Nb pair aδNb408-aδNb107 titrated on Namalwa cells. MFI, median fluorescence intensity; AF488, Alexa Fluor 488; A.U., arbitrary units.

Article Snippet: Anti-IgD Nbs aδNb107 (Addgene 220319), aδNb367 (Addgene 220320), aδNb408 (Addgene 220321) and aδNb571 (Addgene 220322) in pET-15b were codon-optimized for E. coli expression and synthetized by GenScript, with an N-terminal periplasmic leader and a C-terminal TEV cleavage site, followed by a glycine-serine linker and His 6 -tag.

Techniques: Binding Assay, Flow Cytometry, Labeling, Control, Comparison, Construct, Fluorescence

Journal: Scientific Reports

Article Title: Anti-IgD nanobodies as novel tools for studying human IgD biology

doi: 10.1038/s41598-025-09118-4

Figure Lengend Snippet: Characterization of anti-IgD Nb pairs. ( a ) Surface plasmon resonance data showing binding of 25 nM aδNb408 or 25 nM aδNb408-Nb pairs to IgD (HAPPID1) captured by its antigen Phl p 7. RU, resonance units. ( b ) Dynamic light scattering of aδNb408-Nb pairs (assembled using DoubleCatcher) pre-incubated with (or without) IgD-Fc. Hydrodynamic radius was plotted, with the mean shown as a gray bar and the individual data points plotted as black crosses (n = 2). ( c ) Size exclusion chromatogram of aδNb408-Nb pairs post-DLS in complex with IgD-Fc, or IgD-Fc only. Approximate locations of inferred interaction stoichiometries are indicated. 1, unbound Nb pairs or unbound IgD-Fc; 1:1, complex formed by one Nb pair interacting with one molecule of IgD-Fc; 2:1, complex formed by two Nb pairs interacting with one molecule of IgD-Fc; 2:2, complex formed by two Nb pairs interacting with two molecules of IgD-Fc; > , complex formed of more than two Nb pairs and two molecules of IgD-Fc.

Article Snippet: Anti-IgD Nbs aδNb107 (Addgene 220319), aδNb367 (Addgene 220320), aδNb408 (Addgene 220321) and aδNb571 (Addgene 220322) in pET-15b were codon-optimized for E. coli expression and synthetized by GenScript, with an N-terminal periplasmic leader and a C-terminal TEV cleavage site, followed by a glycine-serine linker and His 6 -tag.

Techniques: SPR Assay, Binding Assay, Incubation

Journal: Journal of Pharmaceutical and Biomedical Analysis

Article Title: Forced degradation studies of medroxyprogesterone acetate injectable suspensions (150 mg/ml) with implementation of HPLC, mass spectrometry, and QSAR techniques

doi: 10.1016/j.jpba.2020.113352

Figure Lengend Snippet: Chemical structures for medroxyprogesterone acetate (MPA) and its known impurities . A - 6-hydroxymedroxyprogesterone acetate (6β-hydroxy-6-methyl-3,20-dioxopregn-4-en-17-yl acetate); B – medroxyprogesterone (17-hydroxy-6α-methylpregn-4-ene-3,20-dione); C - 6α,17α-dimethyl-3,17-dioxo- d -homoandrost-4-en-17α-yl acetate; D - 6-epimedroxyprogesterone acetate (6β-methyl-3,20-dioxopregn-4-en-17-yl acetate); E - 6-methylenehydroxyprogesterone acetate (6-methylidene-3,20-dioxopregn-4-en-17-yl acetate); F - 4,5-dihydromedroxyprogesterone acetate (6α-methyl-3,20-dioxo-5β-pregn-17-yl acetate); G - megestrol acetate (6-methyl-3,20-dioxopregna-4,6-dien-17-yl acetate); H - hydroxyprogesterone acetate (3,20-dioxopregn-4-en-17-yl acetate); I - 17β-hydroxy-6α,17α-dimethyl- d -homoandrost-4-en-3,17-dione.

Article Snippet: Medroxyprogesterone acetate for system suitability CRS (contains impurities A, B, C, D, E, G, I) and medroxyprogesterone acetate for peak identification (containing impurity F) were obtained from the European Pharmacopoeia through the European Directorate for the Quality of Medicines and Healthcare (EDQM, Strausbourg, France).

Techniques:

Journal: Journal of Pharmaceutical and Biomedical Analysis

Article Title: Forced degradation studies of medroxyprogesterone acetate injectable suspensions (150 mg/ml) with implementation of HPLC, mass spectrometry, and QSAR techniques

doi: 10.1016/j.jpba.2020.113352

Figure Lengend Snippet: Example chromatogram for Reference Solution A with assignments for respective medroxyprogesterone impurities [ , , ], including relative retention times and +/- 2% RRT range observed for implemented chromatographic conditions.

Article Snippet: Medroxyprogesterone acetate for system suitability CRS (contains impurities A, B, C, D, E, G, I) and medroxyprogesterone acetate for peak identification (containing impurity F) were obtained from the European Pharmacopoeia through the European Directorate for the Quality of Medicines and Healthcare (EDQM, Strausbourg, France).

Techniques: