Journal: Cell

Article Title: Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

doi: 10.1016/j.cell.2021.01.035

Figure Lengend Snippet:

Article Snippet: Five-fold serial dilutions of polyclonal macaque (Molecular Innovations) or human IgG, starting at a concentration of 1 μg/mL, were added to wells containing the coated goat anti-Human IgG λ and κ.

Techniques: Purification, Recombinant, Mass Spectrometry, Sequencing, Ligation, Luciferase, Enzyme-linked Immunospot, Plasmid Preparation, Software, Chromatography, Luminex, Expressing

Journal: EMBO Reports

Article Title: PARP1 condensates differentially partition DNA repair proteins and enhance DNA ligation

doi: 10.1038/s44319-024-00285-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: anti-Mouse IgG, HRP , Cell Signaling Technology , 7076 V.

Techniques: Recombinant, Sequencing, Lambda DNA Preparation, Western Blot, Mutagenesis, Staining, Software

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: Blood was collected from mothers just before or after birth and from neonates birth. A) Transport rates for all IgG subclasses expressed as cord/maternal ratios found at birth. The transport rates differed significantly from each other (P<0.0001), except for IgG2 and IgG3 (not significant), as tested by one-way Anova and Tukey's multiple comparison test. (B–E)IgG subclass 1–4 serum levels were quantified by nephelometry and each pair was plotted on a X axis displaying days of each pregnancy against IgG concentration. Average neonate concentration was significantly higher than in the mother for IgG1 and IgG4 as tested by a paired-T test as shown (child/mother ratio = 1.55 and 1.38, respectively) while averge concentrations for IgG2 and IgG3 were not significantly different in mothers and their children (child/mother ratios not significantly different from 1). One pre-term baby was identified displaying low transport of all IgG (square symbol). (F) Child/mother transport ratio of subclasses IgG2-4 for each pair was plotted relative to the IgG1 transport ratios.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Comparison, Concentration Assay

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: In general, IgG1 is transported better than IgG4, both of which are transported better than IgG2 and IgG3, which have similar transport rates. Two-tailed Pearson correlation revealed a significant correlation for all subclasses, IgG1 R 2 = 0.379, P = 0.0018; IgG2 R 2 = 0.2910, P = 0.0096; IgG3 R 2 = 0.2415, P = 0.0202; IgG4 R 2 = 0.2881, P = 0.0121. Thus, for all subclasses, relatively more IgG was transported at lower maternal IgG.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Two Tailed Test

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: Binding of titrated amounts of soluble human FcRn (62.5–8000 nM) at pH 6.0 to human IgG variants immobilized onto CM5 sensor flow cells. The relative affinity constants derived (KD) are indicated.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Binding Assay, Derivative Assay

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: Results from IgG1 total (A) and IgG2 total (B) were plotted against the sum of IgG1κ and IgG1λ, or IgG2κ and IgG2λ, respectively. The results of regression analysis are indicated in each panel, along with Pearson's correlation.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques:

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: IgG1κ (A), IgG1λ(B) and IgG2κ (C) IgG2λ (D) light chain isotype from sera in were quantified by subclass- and light chain specific ELISA and each mother-child pair was plotted on the x- and y-axis, respectively. A paired t-test revealed no significant difference between the light chains isotypes within each antibody subclass. Average neonate concentration was significantly higher than in the mother for IgG1κ (A) and IgG1λ (B) as indicated in each graph by P values (child/mother ratio = 1.60 and 1.56, respectively) while average concentrations for IgG2 κ and IgG2 λ (C and D) were not significantly different in mothers and their children. (E) Child/mother transport ratio of IgG1λ, IgG2κ and IgG2λ for each pair was plotted relative to IgG1κ transport ratios. While both IgG2 isotypes perform worse than IgG1 when concentration increases, no difference is visible between the IgG2-light chain isotypes.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: (A) Recombinant human IgG2κ and λ in Balb/C mice was injected and measured by total IgG ELISA for a two week period following injection of 200 µg IgG. Calculated half-lives were 7.2±1.48 and 6.4±0.84 days for IgG2κ and IgG2λ. (B) Enrichment of IgG2 κ isoforms was performed as described in Dillon et al 2008. HPLC elution profiles of IgG2 κA and κB structural isomeres on a Dionex ProPac WCX-10 (4.0_250 mm) column are depicted. IgG2κB isoform was generated by incubation of 3 mg/ml IgG2κ in 200 mM Tris buffer at pH 8 with 6 and 1 mM of cysteine and cystamine, respectively. For IgG2κA synthesis 0.9M guanidine hydrochloride (GuHCl) was also added. The samples were kept in the dark and placed at 4°C for 48–72 h. Following incubation the antibody was run on a Zeba spin desalting column (Pierce) for buffer exchange into PBS. (C) The clearance of IgG2λ, IgG2κA, and IgG2κB in Balb/C mice. Calculated half-lives were 4.0±0.58, 5.39±0.85, and 3.7±1.04 days for IgG2κA, IgG2κB and IgG2λ, respectively. (D) Clearance of IgG2κA and IgG2κB in WT and FcγR −/− C57Bl/6 mice. Calculated half-lives were 6.2±2.62, 6.43±1.69, and 7.5±1.89 days for IgG2κA, IgG2κB and IgG2λ, respectively, in WT mice but 1.08±0.28, 1.19±0.23, and 0.7±0.92 days for IgG2κA, IgG2κB and IgG2λ, respectively, in FcRn −/− mice. Graphs in (A, C–D) depict mean and standard deviations of results obtained for 4 mice per group. Half-lives were calculated assuming exponential decay and reported in days ± standard error of means. No significant difference in half-life was detected between the two isotypes.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Recombinant, Injection, Enzyme-linked Immunosorbent Assay, Generated, Incubation, Buffer Exchange

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: (A) The average IgG1 and IgG2 placental transport (maternal/child) ratios were compared according to their light chain isotype. (B) Clearence of IgG1 and IgG2 κ and λ was investigated in humans by collecting blood from hypogammaglobulinemia patients four weeks after an IVIg transfusion. IgG1 and IgG2 light chain isotypes κ and λ were quantified in serum by subclass- and light chain specific ELISA and subclass composition was compared to that found in the IVIg used. No preferential clearance of one light chain isotype was detectable in either IgG subclass.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell

Article Title: Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

doi: 10.1016/j.cell.2021.01.035

Figure Lengend Snippet:

Article Snippet: goat anti-Human IgG λ , Southern Biotech , Cat# 2070-01; RRID: AB_2795749.

Techniques: Purification, Virus, Recombinant, Mass Spectrometry, Sequencing, Emulsion, Adjuvant, Liposomes, Reverse Transcription, Ligation, Luciferase, Enzyme-linked Immunospot, Plasmid Preparation, Software, Chromatography, Luminex, Expressing

Journal: bioRxiv

Article Title: Alloreactivity and autoreactivity converge to support B cell epitope targeting in transplant rejection

doi: 10.1101/2023.03.31.534734

Figure Lengend Snippet: (A) Cells from the transplant nephrectomy specimen and peripheral blood mononuclear cells were stained with fluorescently labeled recombinant HLA tetramer matching the organ transplant donor allele of HLA-A*01:01. Live CD19 + non-naïve (not IgD + CD27 - ) cells that stained double positive with the A*01:01 tetramers were index-sorted for single cell recombinant monoclonal antibody (rmAb) production. (B) Three rmAbs derived from kidney memory B cells were studied for binding to HLA-A*01:01. Binding kinetics are shown, fit as 1:1 Langmuir curves against biotinylated A*01:01 by localized surface plasmon resonance (L-SPR). (C) A plot showing the anti-A*01:01 affinity of all expressed rmAbs. Following kinetic fitting for all rmAbs, anti-A*01:01 equilibrium constants were calculated (KD). Based upon the maximum tested concentration of each rmAb, 1.3uM, a limit of detection for the weakest KD of 1E-5M was inferred. (D) n=52 rmAbs were incubated with 40 different HLA-coated microbeads and stained with an anti-IgG secondary antibody. Geometric mean fluorescence intensities (MFIs) were normalized against a panel of negative (flu-specific) control human rmAbs. Normalized MFIs were log transformed and clustered using Euclidean distance as the similarity measure. All antibodies show anti-A*01:01 binding above the negative threshold value of the assay, and a variety of reactivity patterns with other HLA Class I alleles. (E) rmAb reactivity patterns were further clustered by converting normalized MFIs into binary variables (positive/negative) if the normalized MFI for the antigen was at least 10% of the A*01:01 normalized MFI. Following this transformation, rmAbs were then grouped by patterns of reactivity, testing a null hypothesis that there would be 7 different patterns of reactivity corresponding to the 7 mismatched amino acid residues expressed on the donor A*01:01 as compared to the recipient’s HLA typing. Compared to this null hypothesis, an A*01:01 monospecific pattern of reactivity was observed significantly more often than expected (36.5% vs. 14.3%, p<0.0001). (F) Expanded lineage of B cells depicted using a phylogenetic tree, constructed using Phylip’s DNA parsimony tool, with nodes indicating cell surface phenotype and edges tracing mutations away from an unmutated common ancestor (black node) through inferred mutations (gray nodes) for Lin4, for which related rmAbs displayed an A*01:01 monospecific pattern of reactivity consistent with the immunodominant pattern found in (E) . Data were analyzed using a binomial test and the confidence interval of the proportion was calculated using the hybrid Wilson/Brown method (E) .

Article Snippet: HLA-coated microbeads (OneLambda, ThermoFisher) were incubated with single recombinant monoclonal IgG1 antibodies diluted to 10ug/mL in PBS for 15 minutes.

Techniques: Staining, Labeling, Recombinant, Derivative Assay, Binding Assay, SPR Assay, Concentration Assay, Incubation, Fluorescence, Transformation Assay, Construct

Journal: bioRxiv

Article Title: Alloreactivity and autoreactivity converge to support B cell epitope targeting in transplant rejection

doi: 10.1101/2023.03.31.534734

Figure Lengend Snippet: (A) Heatmap depicting the anti-HLA reactivity patterns of the recipient’s plasma and A*01:01-specific circulating antibody repertoire. The transplant recipient’s plasma (diluted from 1620-fold to 320,000-fold) and A*01:01-specific immunoglobulins (Ig) from plasma that had been affinity purified using an HLA-A*01:01 affinity column (diluted from 5000 ng/mL to 2 ng/mL) were incubated with HLA-coated microbeads, stained with an anti-IgG fluorescent secondary, and anti-HLA geometric mean fluorescence intensity (MFI) values were determined via flow cytometry. MFI 1000 was set as a negative threshold, and MFI values were log10 transformed. (B) Plot comparing the monoclonal repertoire of HLA reactivities to the A*01:01-specific circulating antibody repertoire. The upper plot shows the number of HLA Class I alleles bound by the n=52 rmAbs, where a positive allele is one whose MFI is at least 10% of the A*01:01 MFI for a given rmAb. The bottom plot shows the same MFI dilution data that is depicted in (A) for the A*01:01-affinity purified Ig. (C) Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of a tryptic digest of the A*01:01-affinity purified plasma Ig. The MS-derived peptide amino acid (aa) sequences were used to probe a RepSeq database including all BCR sequences derived from the kidney, blood and rmAb rep-seq libraries (13,466 lineages with >1 one sequence) and an unrelated large RepSeq dataset (323,000 lineages). Exact peptide matches to lineage sequences were determined and members of the lineages were ranked by % aa matched of total. Peptide sequences are plotted by position within VDJ sequence and colored by the frequency of matches across the data set. 68% of the C16 heavy chain VDJ was identified, including two peptide sequence spanning CDR regions that are rare within the sequence database. (D) Lin3, an expanded lineage of alloreactive and autoreactive B cells containing the C16 rmAb from panel (C) , depicted using a phylogenetic tree, constructed using Phylip’s DNA parsimony tool, with nodes indicating cell surface phenotype and edges tracing mutations away from an unmutated common ancestor (black node) through inferred mutations (gray nodes). (E) A plot showing the anti-A*01:01 (alloreactive) and anti-A*24:02 (autoreactive) affinity of n=3 tested rmAbs within Lin3. Following kinetic fitting for all rmAbs, anti-HLA equilibrium constants were calculated (KD). Based upon the maximum tested concentration of each rmAb, 1.3uM, a limit of detection for the weakest KD of 1E-5M was inferred.

Article Snippet: HLA-coated microbeads (OneLambda, ThermoFisher) were incubated with single recombinant monoclonal IgG1 antibodies diluted to 10ug/mL in PBS for 15 minutes.

Techniques: Affinity Purification, Affinity Column, Incubation, Staining, Fluorescence, Flow Cytometry, Transformation Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Sequencing, Construct, Concentration Assay

Journal: eLife

Article Title: Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control

doi: 10.7554/eLife.61389

Figure Lengend Snippet:

Article Snippet: Primary antibodies, sources, and concentrations used were rabbit anti-GFP (Invitrogen) 1:500, rabbit anti-dsRed (Clontech) 1:200, mouse anti dsRed (Clontech) 1:200, mouse anti Hts1B1 (DSHB) 1:20 , mouse anti-γH2Av (DSHB) 1:1,000 , and mouse anti-Orb 4H8 isotype IgG1 (DHSB) 1:500.

Techniques: Sequencing