ANA

    ANA: Anti nuclear antibodies (ANA) is single stand. Important for the differential diagnosis rheumatic diseases.

Indication:

1.      SLE (systemic lupus erythematosus)

Principle: serum sample diluted 1:101 are incubated in the microplates coated with the specific antigen. Patient’s antibodies, if present in the specimen, bind to the antigen. The unbound fraction is washed off in the following step. Afterwards anti-human immunoglobulins conjugated to horseradish peroxides (conjugate) are incubated and react with the antigen-antibody complex of the samples in the microplates.Unbound conjugate is washed off in following step. Addition of MTB substrate generates an enzymatic colorimetric (blue) reaction. Which is stopped by diluted acid (color changes to yellow).the rate of color for mation from the chromogen is function of the amount of conjugate bound to the antigen-antibody complex and this is proportional to the initial concentration of the respective antibodies in the patient sample.

Sample dilute:

1.      Serum 10 ul

2.      Sample buffer 1000 ul

   

   Wash buffer:

1.      dilute wash buffer 20 ml

2.      distiled water           980 ml

Test procedure:

1.      100 ul patients dilute serum in to the designated microwells.

2.      100 ul cut-off calibrator and negative and positive controls in to the designated wells.

3.      Incubate for 30 minutes at 20-32 degree C

4.      Wash 3 times with washing buffer.

5.      100 ul conjugate in to well.

6.      Incubate for 30 minutes at 20-32 C

7.      Wash 3 times with washing buffer

8.      100 ul TMB substrate in to each well.

9.      100 ul stop solution in to each well.

10.  Incubate 5 minutes

11.  Read absorbance at 450 nm within 30 minutes. 

Normal range:

1.      Negative     : 1-12 u/ml

2.      Equivocal   : 12-18 u/ml

3.       Positive     : >18 u/ml