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Dsdna from calf thymus

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DsDNA from calf thymus is a laboratory product that provides double-stranded DNA extracted from calf thymus tissue. It serves as a source of DNA for various research and experimental applications. The product maintains the natural structure and characteristics of dsDNA without further interpretation or extrapolation on its intended use.

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Lab products found in correlation

Maxisorp plate, by Thermo Fisher Scientific (2 mentions) Poly l lysine (pll), by Merck Group (2 mentions) Nucleohistone, by Merck Group (2 mentions) Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions) Alkaline phosphatase conjugated goat anti mouse igg, by Southern Biotech (1 mentions) Yeast rna, by Merck Group (1 mentions) Igg2a, by Southern Biotech (1 mentions) Tmb substrate solution, by Thermo Fisher Scientific (1 mentions) Ap conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)

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Calf thymus dsDNA (28 citations)

6 protocols using dsdna from calf thymus

1

Measuring Anti-dsDNA Antibodies by ELISA

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Levels of anti-dsDNA were measured by ELISA. 20 µg/ml % poly-l-lysine (Sigma-Aldrich) was used to precoat MaxiSorp plates (Nunc). After 2 h at RT, plates were coated with dsDNA from calf thymus (20 µg/ml; Sigma-Aldrich) in TE (pH 7.5) buffer overnight at 4°C. Plates were washed with PBS/0.05% Tween 20 and sera was added in 1/2 serial dilutions starting at 1/100. A serum pool obtained from 9-month-old diseased NZB/W mice served as internal standard. The starting dilution of 1/200 of the NZB/W serum was arbitrarily assigned to 100 relative units. Goat-anti-mouse IgG or goat-anti-mouse IgM, Fc-specific, coupled to horseradish peroxidase was used for detection (Jackson ImmunoResearch).
Weisenburger T., von Neubeck B., Schneider A., Ebert N., Schreyer D., Acs A, & Winkler T.H. (2018). Epistatic Interactions Between Mutations of Deoxyribonuclease 1-Like 3 and the Inhibitory Fc Gamma Receptor IIB Result in Very Early and Massive Autoantibodies Against Double-Stranded DNA. Frontiers in Immunology, 9, 1551.
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2

Enzyme-Linked Immunosorbent Assay for Nucleosome and RNA Detection

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For ELISA, polystyrene plates were coated overnight with poly- L-lysine (Sigma). For anti-nucleosome ELISA, they were then incubated with ethanol-extracted and S1 nuclease-treated dsDNA from calf thymus (Sigma), followed by an incubation with calf thymus histones type II-AS (Sigma). For anti-RNA ELISA, the plates were incubated with yeast RNA (Sigma). Serial dilutions of serum from 1/200 to 1/5400 were added, after blocking with 1% BSA in PBS. Specific Abs were detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology) or IgG2a (Southern Biotechnology). The nucleosome-specific antibody clone PL2–3 and the monoclonal antibody BWR4 were used as standards for the anti-nucleosome and anti-RNA measurements respectively13 (link), 66 . ELISA data were recorded with SpectraMax ELISA Reader and analyzed with DeltaSoft JV (v2.8.11).
Leibler C., John S., Elsner R.A., Thomas K.B., Smita S., Joachim S., Levack R.C., Callahan D., Gordon R., Bastacky S., Fukui R., Miyake K., Gingras S., Nickerson K.M, & Shlomchik M.J. (2022). Genetic Dissection of TLR9 Reveals Complex Regulatory and Cryptic Pro-inflammatory Roles in Murine Lupus. Nature immunology, 23(10), 1457-1469.
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3

Measuring Autoantibody Levels by ELISA

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Serum titers of immunoglobulin subclasses were determined by specific ELISA kits (SouthernBiotech) according to the manufacturer's protocol. To detect anti-dsDNA autoantibodies in sera, high-binding ELISA plates were coated overnight with 2 μg/ml dsDNA from calf thymus (Sigma-Aldrich). Coated plates were blocked with 1% BSA and 0.5% gelatin in TBS for 2 h at room temperature, and diluted samples were incubated overnight at 4°C in TBS with 1% BSA. Bound anti-dsDNA antibodies were detected with AP-conjugated anti-mouse IgG (Jackson ImmunoResearch) and streptavidin-HRP (Bioresearch) followed by TMB substrate solution (eBioscience). Absorbance was measured at 450 nm. Serum titers of anti-ANA antibodies were determined by ANA Hep Screen ELISA kit (Demeditec) according to the manufacturer's protocol.
Zhang H., Hu Q., Zhang M., Yang F., Peng C., Zhang Z, & Huang C. (2019). Bach2 Deficiency Leads to Spontaneous Expansion of IL-4-Producing T Follicular Helper Cells and Autoimmunity. Frontiers in Immunology, 10, 2050.
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4

Quantifying Gut Autoantibodies via ELISA

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Antibody titers against nAgs (nucleohistone and dsDNA) in aforementioned fecal extracts were determined by employing in-house indirect ELISA [3 (link), 5 (link)] with necessary modifications. Briefly, 0.5μg/well of nucleohistone (Sigma-Aldrich) or dsDNA from calf thymus (Sigma-Aldrich) was coated as antigen (carbonate buffer or DNA-coating reagent from Invitrogen respectively), overnight, onto ELISA plate wells. These wells were then incubated with serial dilutions of the fecal extracts (starting at 1:20 dilution) for 2 hours and followed by 1 hour incubation with biotin-linked anti-human IgA, IgA1, and IgA2 antibody. Streptavidin-HRP was added to the well for 30 min, before developing the reaction using TMB/H202 substrate and the plates were read at a wavelength of 450 nm. Highest dilution of the sample that produced an OD value of ≥0.05 above background value was considered as the nAg reactive titer of 100 mg feces. These titer values were then converted to per gram nAg reactivity titer with a multiplication factor of 10.
Nougarède R., Della Seta F., Zarzov P, & Schwob E. (2000). Hierarchy of S-Phase-Promoting Factors: Yeast Dbf4-Cdc7 Kinase Requires Prior S-Phase Cyclin-Dependent Kinase Activation. Molecular and Cellular Biology, 20(11), 3795-3806.
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5

Detecting dsDNA Autoantibodies in Mice

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Detection of dsDNA autoantibodies in mouse sera was performed as described (19 (link)). In Short: 0.01% Poly-L-lysine (Sigma-Alderich) were used to precoat MaxiSorp plates (Nunc). After 2 h at RT, plates were coated with dsDNA from calf thymus (20µg/ml; Sigma-Aldrich) in H20 over night at 4°C. Sera were added in 1/3 serial dilutions starting at 1/20, pooled serum of SLE affected MRL/lpr mice, with a starting dilution of 1/250, served as standard. Goat-anti-mouse IgG (1 mg/ml) coupled to AP was used for detection.
Szumilas N., Corneth O.B., Lehmann C.H., Schmitt H., Cunz S., Cullen J.G., Chu T., Marosan A., Mócsai A., Benes V., Zehn D., Dudziak D., Hendriks R.W, & Nitschke L. (2021). Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections. Frontiers in Immunology, 12, 698420.
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6

Quantification of Autoantibodies and Steroid Hormones

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Levels of antibodies against nucleohistone and dsDNA in mouse sera were determined by ELISA as described in our recent reports [13 (link), 29 (link)]. Briefly, 1.0μg/well of nucleohistone (Sigma-Aldrich) or dsDNA from calf thymus (Sigma-Aldrich) was coated as antigen, overnight, onto ELISA plate wells. Serial dilutions of the sera were made and total IgG and IgG isotypes against these antigens were detected using HRP-conjugated respective antimouse immunoglobulin isotype antibodies (Sigma-Aldrich, eBioscience and Invitrogen). Serum testosterone and 17β-estradiol (estrogen) levels were determined using ELISA and EIA kits from ALPCO and Enzolifesciences respectively.
Johnson B.M., Gaudreau M.C., Gudi R., Brown R., Gilkeson G, & Vasu C. (2020). Gut microbiota differently contributes to intestinal immune phenotype and systemic autoimmune progression in female and male lupus-prone mice. Journal of autoimmunity, 108, 102420.
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