I did a survey for AIV in bird serum by ELISA (coated with NP antigen) and HI (H5 and H9) tests. However, there are samples that give positive results by HI but were negative by ELISA. Is there a relation between them and the presence or absence of replicated or active viruses?
Nope, ELISA results are more trustful, ELISA is more sensitive ..... what is positive by ELISA is the truly positive ones and for HI they may be false positive .... you should run your experiment in duplicates to confirm ......
Another point that, what is positive by ELISA may be other influenza sub-type .... NP is internal gene which is conserved in most influenza sub-types (H1-H16) ... and you used only H5 and H9 and so they may another sub-type emerge (only if you are pretty sure that your technique is totally right
To confirm your HI results .... hold the plate in oblique position and notice the (tears appearance of the RBCs) ..... samples which show tears appearance are usually positive ...
Thanks Ibrahim Thabet
however, I was following OIE manual so no problem for the technique itself but my question is there is a relation between active replication and NP, HA antibodies. In another way, NP positive means virus shedding (I can find it in the sample) and HI positive may be virus release (recent) or old infection
Indeed, I do not think so, how virus shedding occurs without HA spikes ... how can the virus attach to the next bird !! ..... but anyway, you can check for the NP positive samples by RT-PCR, I do believe that you will find them positive either by H5 or H9 primers except if there is other sub-type emerge like H7 (Also, this scenario may be rare because there is no proof of circulation of H7 in Egypt until now)..
Let me tell you about immuno-histochemistry ... I did use NP polyclonal antibody and the samples were positive by NP and also positive by H5 primers using Real-time PCR ... this the reasonable situation .... Influenza viruses are replicating very fast so there is no chance of delayed virion packing (at which you can find only NP and you can not HA) as you assume ...
Another scenario .... if your samples were from vaccinated birds .... so they may harbour antibodies against HA (which indicate vaccination) and not NP (which indicate infection because farms usually use inactivated vaccines) ..... in that case you designed your ELISA for checking NS-1 protien because it appears only in infection and not vaccination OR you should take two serum samples between them 15 days and do ELISA (against HA) or HI and compare the titer ... if the titer were the same this means vaccination ... if there was change in titers by significant folds this means infection ....
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