Irregular results in hemagglutinin results?

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MOHIT
MOHIT's picture
Irregular results in hemagglutinin results?

I was performing the hemagllutinin assay in the labortary.I had all the reagents properlly made and there was proper procedure followed during the assay but i got very irregular results.There was earlier mat formation and then in the miidle of the microtiter plate there was button formation and again the mat formation.can anyone could help me out of it?

Sami Tuomivaara
Sami Tuomivaara's picture
MOHIT,

MOHIT,

I have couple of things to think about:

One explanation for non-monotonous hemagglutination curve is if you have extremely high virus titer in the high dilution end, it can prevent hemagglutination because all the RBCs are covered with virus particles, there is no RBC-virus-RBC links formed and hence no hemagglutination (false negative result). Upon slight dilution, you get proper ratio of virus and RBCs and get hemagglutination (positive result) and upon more dilution, you get negative again since there is not enough virus particles for hemagglutination...

Also, remember that the RBCs form a suspension, not a homogenous solution, in the serum. It is actually quite easy to pipet variable amount of RBCs to different tubes or wells if care is not taken to mix the RBS suspension well before pipetting.

Hope this helps even a bit...

Cheers,

MOHIT
MOHIT's picture
Thanks for your reply.but we

Thanks for your reply.but we were carring the experiment with the bacteria and again when we performed the same we got a systemic array of buttons and then the mat.we did in duplicates and in one set we get all the buttons.i was told that "Those buttons laso give positive results as we have to observe for the agglutination and the fine structure appear around the buttons which are the bacterial structures responsible for the outgrowth"is this true as it created confusion with me because earlier it was told that the buttons are the negative resuts.

Sami Tuomivaara
Sami Tuomivaara's picture
MOHIT,

MOHIT,

Could you please describe your results very specifically, from your desription, I'm not sure in which concentration of bacteria the mats and buttons appear... Please specify whether you get mat or button (or something else) in each of the concentrations of bacteria in your duplicate experiments.

The button _should_ be negative result, and mat a positive, but like you said, there are more than two possible morphologies of the result and the interpretation might be difficult.

Cheers,

MOHIT
MOHIT's picture
We had 25 µl of 2% RBC

We had 25 µl of 2% RBC suspension in PBS which was mixed with the 25  µl of log phase bacterial culture of e.coli which had 108 cfu/ml.The RBC suspension was added in the microtiter plate and PBS with mannose was added in one row and PBS without mannose added in other row.Then the bacterial culture was added and serial dilutions made from 1:2 to 1:128 such that 25 µl was removed from the last well.The results that were obtained on the microtiter plate had a systemic array of buttons and then the mat.One thing that i observed was that in the initial dilutions the solution was somewhat clear but as the dilution increased the solution had some reddish tinge very light one.The buttons were like dark in the centre and then around them there was a circular zone and we were told that "There are structures which have linkage of button to the outer zone.so the test is positive" But i was able to agree with the same.