I am doing HI Test for NDV and Avian Influenza Viruses but before going to start I first tested the serum whether any reactivity exists or not with 1% washed Chicken RBC. but I found that some serum show reactivity and even hemagglutinate the chicken RBC. My question is, that apart from heat treatment is there any other simple treatment so that to inhibit the serum hemagglutination activity because I heated it at 56 C for 30 minutes but it won't work. Please guide.
I am not far from the protcol which include first inactivation to destroy non specific inhibitors second get rid from low concentration of agglutinins by adsorption with chicken RBC. I think the question not need the procedure Thanks
First of all you must do inactivation for non specific inhibitors by using water bath 56 for 30 minutes.Thanks
I got this from FAO manual.
Natural agglutinins
Some chicken sera contain substances that will agglutinate chicken red blood cells. The settling pattern of the agglutinated cells is similar to that produced by Newcastle disease virus. These natural agglutinins are present in low concentration. The control serum well will indicate the presence of natural agglutinins.
Adsorption of natural agglutinins
If natural agglutinins are present in a serum sample, they can be removed by adsorption with chicken red blood cells. The serum can then be retested in the HI test.
Materials
- Suspension of 10 percent washed chicken red blood cells.
- Microfuge (Eppendorf) tube
- Micropipette, 200 mL to 1 000 mL and tips
- Discard tray
- Microfuge centrifuge
- Serum samples
Method
1. Place 200 mL of the 10 percent washed red blood cells into a microfuge tube.
2. Centrifuge for 15 seconds.
3. Remove the supernatant.
4. Shake the serum sample and remove 500 mL of serum. Some samples may contain less volume. Use a new tip for each sample.
5. Add the serum to the red blood cells in the microfuge tube. Mix gently.
6. Stand for not less than 30 minutes at 4°C.
7. Centrifuge for 15 seconds.
8. Remove the serum (supernatant) immediately and transfer to a clean microfuge tube.
9. Store the adsorbed sera at 4°C overnight or at -20°C for longer periods.
Removal non specific inhibitors by inactivation of the sera and get rid from the low concentration agglutinin by adsorption with chickenRBC ,then the sera can be rested in the HI test.
I agree with comments from Hanif Ur Rahman and you should follow the protocol. He mentioned about chicken sera, I think, by mistake. Some human sera also have non-specific agglutinins, which should be removed using the protocol described by Hanif Ur Rahman.
I am not far from the protcol which include first inactivation to destroy non specific inhibitors second get rid from low concentration of agglutinins by adsorption with chicken RBC. I think the question not need the procedure Thanks
For colleaques virology expert according to the FAO protcol to solve the agglutinins in the sera which interfere with Hi we must apply the following: A,treatment with recrptors destroying enzyme(RDE)by dilution the serum1/4with RDE then incubate overnight at 37 mixture both srum and RDE.,B,Inactvation at waterbath 56 temperature for 30 minutes to get rid from non specific inhibitors .N.B,All operations dealing with serm collection and seperation must be under aseptic technique. Thanks.
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