msu crystals lot 1  (Merck KGaA)

Journal: Scientific Reports

Article Title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals

doi: 10.1038/s41598-022-08311-z

Figure Lengend Snippet: Natural IgM binds to MSU and CPPD crystals. ( a ) Serum proteins bound to each indicated particle were analyzed by LC–MS. Fraction of each antibody isotype bound to each indicated particle is shown. Data from 2 individual serum samples is depicted. In one serum sample, IgA2 was not detected; therefore, only one data point is shown. ( b ) Fraction of immunoglobulin isotypes bound to MSU or t-CPPD crystals from 14 normal human serum samples quantified from Western blot analysis. ND = not detected. Raw data (blot images) is shown in Supplementary Fig. . ( c ) Individual normal human sera (NHS, including samples from B) or IgM/IgA-deficient serum samples were incubated with the indicated particles, and bound IgM and IgG were detected using anti-IgM PE or anti-IgG PE, respectively. Median fluorescence intensity (MFI) of the particles was normalized by subtracting the MFI of the negative control (FBS). Means of the NHS and IgM/IgA-deficient serum samples were compared by an unpaired t-test. ( d ) Binding of purified polyclonal (poly.) or monoclonal (mono.) IgM to three distinct preparations of MSU crystals in IgM/IgA-def. serum or HBSS + 10% BSA. ( e ) Pooled human serum collected either from healthy adults or from human cord blood was incubated with the indicated particles, and bound IgM was detected as in (c). Values for each particle preparation and the mean are shown. ( f ) MSU (lot 2), silica (SiO 2 ), or calcium carbonate (CaCO 3 ) crystals were incubated in FBS or 4 normal human pool serum samples. Bound human IgM was detected as in (c) and MFI for each serum sample is shown including the negative controls (FBS).

Article Snippet: MSU crystals : Lot 1 (small; mean length: 9.2 ± 4.4 μm): 10 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 10 mM NaOH.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Incubation, Fluorescence, Negative Control, Binding Assay, Purification, Calcium Carbonate

Journal: Scientific Reports

Article Title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals

doi: 10.1038/s41598-022-08311-z

Figure Lengend Snippet: Polyclonal IgM activates complement in the presence of MSU crystals. IgM/IgA-deficient serum obtained from three individuals was depleted of CRP and then reconstituted with polyclonal IgM, IgA, or monoclonal IgM at 0.4 mg/ml. Serum samples were incubated for 30 min at 37 °C with vehicle or MSU (lot 1, 20 mg/ml). The concentrations of anaphylatoxins C4a, C3a, and C5a are shown. Lines represent the means. p values for comparisons of the means were calculated using paired t-test. Arrows above the graphs indicate the sequence of production of C4, C3a, and C5a in the three main complement pathways.

Article Snippet: MSU crystals : Lot 1 (small; mean length: 9.2 ± 4.4 μm): 10 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 10 mM NaOH.

Techniques: Incubation, Sequencing

Journal: Scientific Reports

Article Title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals

doi: 10.1038/s41598-022-08311-z

Figure Lengend Snippet: Non-redundant role of CRP in complement activation by MSU crystals. Four pooled serum samples obtained from healthy individuals and 11 IgM/IgA-deficient serum samples obtained from CVID patients were depleted of residual CRP and reconstituted with CRP (30 µg/ml) or vehicle. Sera were incubated with either MSU lot 1 ( a ) or t-CPPD ( b ) for 30 min at 37 °C. Concentrations of soluble complement system activation products C4a, C3a, C5a, and sC5b-9 in the supernatant are shown. Two serum samples from CVID patients that showed early classical pathway activation are displayed separately (IgM/IgA-def.*). Lines represent the means. For comparison of serum samples with or without CRP, p values were calculated by paired t-test; for comparison of IgM/IgA-sufficient and IgM/IgA-deficient serum samples, an unpaired t-test was used.

Article Snippet: MSU crystals : Lot 1 (small; mean length: 9.2 ± 4.4 μm): 10 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 10 mM NaOH.

Techniques: Activation Assay, Incubation, Comparison

Journal: Scientific Reports

Article Title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals

doi: 10.1038/s41598-022-08311-z

Figure Lengend Snippet: CRP drives anaphylatoxin production in the presence of MSU crystals. IgM/IgA-deficient serum obtained from 3 individuals with CVID was depleted of CRP and then reconstituted with vehicle or CRP at 10 or 30 µg/ml. The serum samples were incubated for 30 min with nothing, MSU lot 2, or cholesterol crystals. The concentrations of anaphylatoxins C4a, C3a, and C5a in the serum after the incubation are shown. p values are shown for the comparison of the means of vehicle, 10 µg/ml CRP, and 30 µg/ml CRP as determined by one-way repeated-measures ANOVA.

Article Snippet: MSU crystals : Lot 1 (small; mean length: 9.2 ± 4.4 μm): 10 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 10 mM NaOH.

Techniques: Incubation, Comparison