ELISA/assay

Anti-MOG (35-55) IgG ELISA Kit

DEIA6141

96T

Anti-MOG (35-55) IgG ELISA Kit

MOG

Anti-MOG (35-55) IgG ELISA Kit

iELISA

MOG

Mouse, Rat

Quantitative

Serum

96T

Kit is shipped on blue ice. Store all kit components at 2-8°C for up to 12 months.

MOG

Mouse, Rat

MOG; myelin oligodendrocyte glycoprotein; myelin-oligodendrocyte glycoprotein; MOG Ig-AluB; MOG alpha-5; MOGIG2; NRCLP7; MGC26137

This kit is optimized to detect mouse/rat anti-MOG (35-55) IgG.

Myelin oligodendrocyte glycoprotein (MOG) is a member of the immunoglobulin superfamily and is expressed exclusively in the central nervous system. MOG (35-55) is able to induce autoantibody production and relapsing-remitting neurological disease causing extensive plaque-like demyelination. Autoantibody response to MOG (35-55) has been observed in multiple sclerosis (MS) patients and MOG (35-55)-induced experimental autoimmune encephalomyelitis (EAE) C57/BL6 mice and Lewis rats. The Anti-MOG (35-55) IgG ELISA Kit (mouse/rat) provides a convenient and quantitative assay for anti-MOG (35-55) autoantibody in mouse and rat. This kit is useful to researchers for determining the amount of anti-MOG (35-55) antibody present, and can help provide information on the role it plays in the development and treatment of EAE, an animal model for MS pathogenesis.

Wells are pre-coated with MOG (35-55) peptide and pre-blocked with BSA. The amount of anti-MOG (35-55) IgG in serum or cerebrospinal fluid is quantified using ELISA. Ample materials and reagents are provided to perform 96 assays.

Component A: MOG (35-55) coated and BSA blocked 8-well strips, 12 strips Component B: Mouse anti-MOG (35-55) IgG standard, 110 μl (5 μg/ml) Component C: 1× Sample Dilution Buffer, 30 ml Component D: 10× Wash Buffer, 50 ml Component E: TMB color substrate solution, 10 ml Component F: Stop Solution, 10 ml Component G: Secondary antibody, Goat anti-Mouse IgG-HRP, 30 μl Component H: Secondary antibody, Goat anti-Rat IgG-HRP, 30 μl Component J: Rat anti-MOG (35-55) IgG standard, 110 μl (5 μg/ml)

1. Microplate reader: Capable of reading absorbance at 450 nm 2. Rocking platform or shaker 3. Strip ejector (to eject strips for future assay if not all strips are used in one experiment) 4. Computer software: Capable of plotting Four Parameter Logistic Curve Fit (4-PL) (optional)

Please Note: 1. Allow kit components to warm up to room temperature before starting the assay 2. Spin down all components with volume less than 100 μl before use 3. Mix well 10× Washing Buffer before diluting to dissolve any precipitated salt 4. More Sample Dilution Buffer can be made by adding 1% BSA into 1× Wash Buffer Procedure: 1. Establish dilution range of serum samples: Serial dilutions of serum samples can start from 1:1k, 1:5k, 1:25k, 1:125k. Use 1X Sample Dilution Buffer (Component C) to dilute samples and standards (an example is shown in Table 1). Depending on the amount of antibody present, the dilution range can be further adjusted. 2. Arrange and label strips (Component A) based on the number of wells with standard and samples. An example is shown in Table 1. Although diluted standard and samples can be run as single points, duplicates are recommended. 3. Depending on the animal model used, dilute mouse or rat anti-MOG (35-55) IgG standard (Component B or J) in 1X Sample Dilution Buffer (Component C) according to the Table 2. 4. Add 100 μl of the diluted standards into wells (A1,2-G1,2 for duplicate run). Add 100 μl of 1× Sample Dilution Buffer (Component C) as a blank into wells H1,2. 5. Add diluted samples into appropriate wells (depends on the number of samples to be tested). After adding the standards and samples to the wells, cover the plate and incubate at room temperature for 60 min with gentle shaking. 6. Prepare 1× working wash buffer by diluting the 10× Wash Buffer (Component D) with DI H 2 O. 7. Wash wells five times at 200 μl/well of 1× washing buffer. Pat dry. 8. Dilute goat anti-mouse IgG-HRP (Component G) secondary antibody (2 nd Ab) with Sample Dilution Buffer (Component C) if quantifying mouse samples or/and goat anti-rat IgG-HRP (Component H) secondary antibody if quantifying rat samples. Secondary antibody working solution is 1:2,000 dilution. Add 100 μl of the diluted 2 nd Ab into each well and incubate plate at room temperature for 45-60 min with gentle shaking. 9. Wash wells five times with 200 μl per well of 1× washing buffer. Pat dry. Clean the outside bottom of the wells with lens paper if necessary before the next step (this ensures accurate absorbance reading). 10. Add 100 μl of the TMB color substrate solution (Component E) into each well. Tap plate gently and incubate at room temperature until blue gradient is clearly observed across the wells (1-15 min depending on the secondary antibody dilution). It may be necessary to adjust color development time so that absorbance values fall within the detection range. 11. Add 50 μl of the Stop Solution (Component F) into each well and tap plate gently (blue color will turn to yellow). Measure absorbance (OD) at 450 nm using a microplate absorbance reader within 20 minutes after adding stop solution.

1. Determine the average values (if replicates are used) for the standard and sample absorbance readings. Plot calibration curve using Four Parameter Logistic (4-PL) curve-fit. R 2 should be higher than 0.99. There should be at least 5 serially diluted standard concentrations in the calculation to ensure statistical significance. 2. Choose absorbance values for the samples that are within the range used in the standard curve, and calculate the concentration of anti-MOG (35-55) IgG using 4-PL curve-fit. 3. Example of calculation of anti-MOG (35-55) IgG concentrations: Please note, new standard curve must be generated each time the assay is run. Note: Columns 1 and 2 are duplicate mouse anti-MOG(35-55) IgG standards 500, 250, 125, 62.5, 31.25, 15.625, 7.8, and 0 ng/ml (Row A ~ H). Mouse Samples: Sample-1, 3A-D and 4A-D; Sample-2, 5A-D and 6A-D; Sample-3, 3E-H and 4E-H; Sample-4, 5E-H and 6E-H (at 1:1k, 5k, 25k, and 125k dilution in duplicates). Columns 7 and 8 are duplicate rat anti-MOG(35-55) IgG standards 500, 250, 125, 62.5, 31.25, 15.625, 7.8, and 0 ng/ml (Row A ~ H). Rat Samples: Sample-1, 9A-D and 10A-D; Sample-2, 11A-D and 12A-D; Sample-3, 9E-H and 10E-H; Sample-4, 11E-H and 12E-H (at 1:1k, 5k, 25k, and 125k dilution in duplicates). Values in bold were used for calculations. Four-parameter logistic curve-fit (4-PL) based on the average absorbance reading values: From 4-PL curve-fit data table (not shown) generated by computer software the following anti-MOG (35-55) concentrations for mouse samples were obtained (based on the average absorbance readings): From 4-PL curve-fit data table (not shown) generated by computer software the following anti-MOG (35-55) concentrations for rat samples were obtained (based on the average absorbance readings):

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet