1Using ChonBlock™ for Anti-Antigen Antibody ELISAs
For Research Use Only - Not Human or Therapeutic Use

1.

Coat Plates with Antigen: Dissolve antigen at 5 - 10 µg/ml in PBS with 0.05% Azide. Add 100 µl of the antigen solution to ELISA
plates and incubate at 4°C overnight. Wash the antigen-coated plates with endotoxin-free distilled water 3 times using a wash bottlewith manifold. Empty the plate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

2.

Add Blocking Buffer: Add 100 µl of the ChonBlockTM Blocking/Sample Dilution Buffer to each well and incubate for 1 hour at room
temperature.

3.

Prepare Sample Dilutions: Dilute 10 µl of serum sample with 990 µl of ChonBlockTM Blocking/Sample Dilution Buffer (1:100 dilution)and keep it as a stock solution for future assays. Then, further dilute the sample with ChonBlockTM Blocking/Sample Dilution Buffer
depending on the antibody levels. For example, take 200 µl of the sample stock solution and mix with 200 µl of ChonBlock TM
Blocking/Sample Dilution Buffer to make a 1:200 dilution.
NOTE: Chondrex, Inc. recommends running a preliminary assay using various dilutions of sera (1:200, 1:1,000, 1:5,000) in order todetermine the optimal dilution of the samples, especially before assaying a large number of samples.

4.

Wash: Wash the plate with a wash buffer (PBS with 0.05% Tween 20) at least 3 times using a wash bottle with manifold or an automatedplate washer. Empty the plate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

5.

Add Samples: Add 100 µl of ChonBlockTM Blocking/Sample Dilution Buffer (blank) and diluted samples to wells in duplicate according
to the desired layout. Incubate at room temperature for 2 hours.

6.

Wash: Wash the plate with the wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty
the plate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

7.

Add Detection Antibody: Dilute appropriate peroxidase-conjugated detection antibodies in 10 ml of ChonBlockTM Detection Antibody
Dilution Buffer. Add 100 µl of the detection antibody solution to each well and incubate at room temperature for 1 hour.
NOTE: Chondrex, Inc. recommends running preliminary assays using a positive control to optimize the detection antibody concentration.

8.

Wash: Wash the plate with the wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty
the plate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

9.

Add TMB: Prepare TMB solution just prior to use. Add 100 µl of the TMB solution to all wells immediately after washing the plate andincubate at room temperature for 10 - 30 minutes.

10. Stop: Add 50 ml of 2N sulfuric acid (Stop Solution) to each well.
11. Read Plate: Read the OD values at 450 nm (a 630 nm filter can be used as a reference).

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Phone: 425.702.6365 or 888.246.6373
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