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Product name : HOSu
Catalog number : 1011
Supplier name : Sceti K.K.
Data sheet: Ask more or other datasheet now !
More Details about
This kit is designed to assay type I and type II collagen antibodies in mouse sera. Chondrex’s ELISA systems incorporate unique blocking agents to reduce non-specific reactions. These agents reduce the background levels by inhibiting the hydrophobic binding of immunoreactive serum components in sample specimens onto plastic surfaces. Various species of type I and type II collagen-coated strips are available as shown on the right. This ELISA kit contains enough material to run two plates on two separate occasions (see assay procedure).
Heterologous type II collagen is widely used as an immunogen for the collagen-induced arthritis (CIA) model. In CIA-susceptible mice, the serum antibody levels to the type II collagen used for immunization are very high. Futhermore, these antibodies cross-react to various species of type II collagen including autologous type II collagen, due to the conserved amino acid sequences of type II collagen.
Importantly, although type I collagen shares the same amino acid sequences with type II collagen by more than 80%, it is not capable of inducing autoimmune-mediated diseases. This indicates that epitope specificity of antibodies and T-cells are important for establishing autoimmunity and subsequent development of autoimmune diseases. Therefore, type I collagen might be useful as a control for studying the pathogenesis of autoimmune mediated arthritis, and B and T-cell epitope specificity.
NOTES BEFORE USING ASSAY
Note 1: It is recommended that the standard and samples be run in duplicate.
Note 2: Partially used reagents may be kept at –20°C.
Note 3: Crystals may form in the 20X wash buffer when stored at cold temperatures. If crystals have formed, it is necessary to warm the wash buffer by placing the bottle in warm water until crystals have dissolved completely.
Note 4: Measure exact volume of buffers using a serological pipette prior to diluting. Extra buffer is provided.
1. Dilute Wash Buffer: Dilute 50 mL of 20X wash buffer in 950 mL of distilled water (1X wash buffer). Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
2. Add Blocking Buffer: Add 100 mL of Blocking Buffer (Solution A) to all wells. Incubate for 1 hour at room temperature.
3. Prepare Standard Dilutions: Dissolve one vial of standard (20 units/vial) in 1.25 mL of Sample/Standard Dilution Buffer (Solution B) to make 16 units/mL solution. Prepare serial dilutions of the standard by mixing 250 mL of 16 units/mL standard with 250 mL of Solution B - 8 units/mL . Then repeat this procedure to make five more serial dilutions of standard - 4, 2, 1, 0.5 and 0.25 units/mL
solutions. The 16 units/mL standard may be stored at -20°C for use in a second assay. We recommend making fresh serial dilutions for each assay.
4. Prepare Sample Dilutions: If necessary, centrifuge serum samples at 10,000 rpm at room temperature for 3 minutes to remove insoluble materials and lipids. Dilute samples 1:1000 or more with Solution B. For example, dilute 10 mL of sample with 0.99 mL of Solution B (1:100). Keep this as a stock solution for future assays. If it is necessary to assay antibodies at a low dilution of less than 1:200 due to low antibody levels, please contact Chondrex Customer Service. In general, the recommended dilution of CIA mouse sera collected at 4 weeks is 1:10,000 and 6-8 weeks is 1:20,000.
5. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
6. Add Standards and Samples: Add 100 mL of standards, Solution B (blank) and samples to wells in duplicate. Incubate at 4°C overnight.
7. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
8. Add Secondary Antibody: Dissolve one vial of secondary antibody in 10 mL Secondary Antibody Dilution Buffer (Solution C).
Add 100 mL of secondary antibody solution to each well and incubate at room temperature for 2 hours.
9. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
10. OPD: Dissolve one vial of OPD in 10 mL of OPD Dilution Buffer just prior to use. Add 100 mL of OPD solution to each well immediately after washing the plate. Incubate for 30 minutes at room temperature.
11 . Stop: Add 50 mL of 2N sulfuric acid (Stop Solution) to each well.
12. Read Plate: Read the OD values at 490 nm. If the OD values of samples are greater than the OD values of the highest standard, re-assay the samples at a higher dilution. A 630 nm filter can be used as a reference.
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