Procedure

PROCEDURE

  • Solid phase matrix
    • Nitrocellulose membrane (NCM) is solid phase matrix conveniently used in the Dot Immunobinding assays.
    • The membrane of 0.22µm or 0.45 µm pore size is commonly used..
    • The membranes are cut into 5x10 cm sizes for testing about 15 to 20 samples. Rough squares are marked with pencil for taking samples.
  • Dotting of Antigen
    • Approximately 0.5-1 µl of antigen is deposited onto the nitrocellulose membrane with help of micropipette.
    • The amount of antigen in term of volume to be deposited on to nitrocellulose membrane depends upon the concentration of protein or other antigens present in solution, is important to achieve desired antigen antibody reaction.  It can be determined by checkerboard titration.
    • The NCM is air dried and kept in a vacuum oven at 80◦ C for 1 hour to fix the antigen to the NCM.
    • The antigen dotted nitrocellulose membrane can be stored for a few months at 4-8◦ c without appreciable loss in antigen activity.
  • Blocking
    • Subsequent to dotting of antigen, blocking of unsaturated sites on the Nitrocellulose membrane is an essential to prevent nonspecific adsorption of the immunological reagents.
    • The unsaturated sites are blocked by immersing the NCM in a 5% dried milk powder in PBST and incubated at 37◦ C for 1 hr.
    • The NCM  is washed in PBST. The NCM immersed in washing solution in a container by holding them in hand from opposite end to membrane and then washing them by quick backward and forward movement for three minute. This process is repeated three times using three different containers.  
  • Incubation with serum sample
    • The antigen dotted NCM is then incubated with 1:100 diluted suspected serum sample for anigen-antibody reaction at 37◦ C for 1 hour.
    • The NCM  is washed in PBST as above.
    • This allows separation of bound antigen or antibody from that of free antigen or non reactive antibody as the case may be.
  • Incubation with conjugate
    • The NCM is incubated with anti species specific immunoglobulins conjugated to an enzyme Horse radish peroxide as conjugate at optimum dilution for 1 hour at 37◦ C
    • Determination of optimum dilution of the conjugate to be used in the assay is very critical because an inappropriate dilution could result in higher background reaction or reduce the sensitivity of the test. It can be obtained using checker board titration.
    • At the end of incubation the NCM is washed three times in PBST each of five minutes.
    • The other enzyme used to prepare conjugate is Alkaline phosphatase.
  • Incubation with substrate solution
    • This is the final step in which specific antigen – antibody reaction is visualized.
    • The substrate solution Diamino benzidine (DAB) is sensitive substrate, yielding a brown colour a the site of antigen deposition.
    • The Nitrocellulose membrane is immersed in the substrate solution, Diamino benzidine (DAB) at room temperature for the development of colour. It takes only few minutes.
    • To stop the reaction, the membrane is washed with distilled water.
    • The color developed nitrocellulose membrane is washed , air dried and can be stored indefinitely in a sealed container.
    • The draw back of this substrate is its over sensitiveness as it reacts very fast so that it is very easy to over develop resulting in high background.
    • The other substrate is Chloronaphthol , gives an intense blue – black colour product .
  • Analysis
    • In positive cases, a brown spot appears at the site of application of sample, the intensity of the color is proportional to the concentration of the antigen.
Last modified: Sunday, 25 September 2011, 10:01 AM