Mouse Anti-dsDNA IgG

Objective: IgG anti-dsDNA

Reactivity: Mouse

Detection method: Colorimetric

Detection range: 0.312-20 ng / ml

Lower limit of detection

0.312 ng / ml

Application: ELISA

Objective

For the quantitative determination of mouse anti-double-stranded DNA (IgG) antibody concentrations in serum, plasma.

Example type: Serum, Plasma

Analytical method: Quantitative

Specificity

This assay has high sensitivity and excellent specificity for the detection of mouse anti-double-stranded DNA (IgG) antibodies.

Cross-reactivity

Limited by current knowledge and skills, we are unable to complete the detection of cross-reactivity between the target antigen and all analogues from other species. Therefore, it is possible that there is still a cross-reaction.

Sensitivity: 0.078 ng / ml

Components 

  • Assay plate (12 × 8 coated microwells)
  • Standard (lyophilized)
  • Biotin-antibody (100 × concentrate)
  • HRP-avidin (100 × concentrate)
  • Biotin-antibody diluent
  • HRP-avidin diluent
  • Sample diluent
  • Wash buffer (25 × concentrates)
  • TMB substrate
  • Stop solution
  • Adhesive strip (for 96 wells)
  • Manual

Material not included

  1. Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set to 540 nm or 570 nm.
  2. An incubator that can provide stable incubation conditions up to 37 ° C ± 0.5 ° C.
  3. Jet bottle, multi-dispenser or automatic microplate washer.
  4. Absorbent paper to dry the microtitre plate.
  5. Graduated cylinders of 100 ml and 500 ml.
  6. Deionized or distilled water.
  7. Pipettes and pipette tips.
  8. Test tubes for dilution.

Application notes

  • The supplier is only responsible for the kit itself, but not for the samples consumed during the test. The user must calculate the possible number of samples used throughout the test. Please reserve enough samples in advance.
  • Samples to be used within 5 days can be stored at 2-8 ° C; otherwise, samples should be stored at -20 ° C (≤ 1 month) or -80 ° C (≤ 2 months) to avoid loss of bioactivity and contamination.
  • Severely hemolyzed specimens are not suitable for use in this assay.
  • If the samples are not indicated in the manual, a preliminary experiment is necessary to determine the validity of the kit.
  • Predict concentration before testing. If the values ​​for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
  • Cell or tissue extraction samples prepared with Chemical Lysis Buffer may produce unexpected ELISA results due to the impact of certain chemicals.
  • Due to the potential for a mismatch between antigens from another resource and the antibodies used in kits from this vendor (for example, the antibody targets the conformational epitope rather than the linear epitope), the products from this vendor may not recognize some native or recombinant proteins from other manufacturers.
  • Due to the influence of factors including cell viability, cell number, and cell sampling time, the kit may not recognize cell culture supernatant samples.
  • Fresh samples without prolonged storage are recommended for testing. Otherwise, degradation and denaturation of proteins can occur in those samples and ultimately lead to incorrect results.

Test time: 1 – 4.5 h

Plate: Precoated

Protocol

The microtiter plate provided in this kit has been pre-coated with an anti-mouse IgG antibody. Standards or samples are then added to the appropriate microtiter plate wells with biotin-conjugated dsDNA and horseradish peroxidase (HRP) conjugated avidin is added to each well of the microplate and incubated. Next, a TMB substrate solution (3,3 ‘, 5,5’ tetramethylbenzidine) is added to each well.

Only those wells containing anti-mouse IgG antibodies, biotin-conjugated dsDNA, and enzyme-conjugated avidin will exhibit a colour change. The enzyme-substrate reaction is terminated by adding a sulfuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. Next, the concentration of anti-double-stranded DNA (IgG) antibody in the samples is determined by comparing the O.D. of the samples to the standard curve.

Sample collection

Serum: Use a Serum Separator Tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4 ° C before centrifuging for 15 minutes at 1000 × g. Remove serum and analyze immediately or aliquot and store samples at -20 ° C or -80 ° C. Avoid repeated freeze-thaw cycles.

Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2-8 ° C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20 ° C or -80 ° C. Avoid repeated freeze-thaw cycles.

Tissue homogenates: Rinse 100 mg of tissue with 1 × PBS, homogenize in 1 ml of 1 × PBS and store overnight at -20 ° C. After two freeze-thaw cycles to disrupt cell membranes, centrifuge the homogenates for 5 minutes at 5000 × g, 2-8 ° C. Remove and analyze the supernatant immediately. Alternatively, aliquot and store samples at -20 ° C or -80 ° C. Centrifuge the sample again after thawing before testing. Avoid repeated freeze-thaw cycles.

Results calculation

Average the duplicate readings for each standard and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve fit (4-PL). Alternatively, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best-fit curve through the points on the graph. Data can be linearized by plotting the log of the target antigen concentration versus the log of the O.D. and the line of best fit can be determined by regression analysis. This procedure will produce an adequate but less precise fit to the data.

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