Hello everybody,
I am working with hemagglutination inhibition test and results I've got are quiet unusual. I'm wondering, why could this happened.
The test I'm trying to do is lectin hemagglutinating activity inhibition with various carbohydrates. I dilute carbohydrate samples from 400 mM to 3,125 mM in twofold serial dilutions, add equal amount (and conc) 50 ul of my sample, incubate for 1 h at 22 C. After incubation I pour 50 ul of 2 % rabbit red blood cells suspension and wait for 2 hours to see the result. After all I get hemagglutinating activity with higher carbohydrate content and inhibition with lower carbohydrate concentration. It should be opposite as mentioned in literature.
Maybe somebody had the same problem and know how to solve it? Or have some ideas, what I am doing wrong there?
Thank you in advance.
I believe the concentration of sugar used is quite high (3.1M), as pointed out by Adam you may observe such agglutination (clumping) even with out adding lectins !!!.
Try to repeat the experiment with relatively lower concentration of sugars. Make sure to include proper controls as well.
Thank you for an ideas, @Adam B Shapiro and @Saravanan Kumar!
Gabriele Balciunaite Muziene I know I am terribly late reading this discussion. Since I am also working with plant lectins and I have faced the same problem as you had mentioned with regard to carbohydrate inhibition assay, I was very keen in knowing if you were able to solve the issue? I have also obtained similar results with inhibition assays wherein at lower sugar concentration (Glucose and mannose in my case) visible inhibition was seen. The stock sugar used was 0.2M(200mM).. Kindly enlighten me as to how you were able to solve the issue.
Awaiting your kind reply
I agree with previous answers - you need use more carbohydrate concentration. I used 0,6 M of carbohydrates. Try use this concentration
Hello, can I know how to calculate the total activity of lectin from hemagglutination??
thank you
The reason for agglutinine activity, since you have used undiluted sample (lectin contains), that's why you got HA activity even after you add sugar. Before start to perform HA inhibition, you must dilute your sample until you get HA titer value to 4 (2 wells only). Moreover, you used high concentration of sugar, so just use only about 200 mM of respective sugars you wanted to check HA inhibition test.
All the best.
Our primary research focuses on lectins. A late reply but could be useful though. Instead of going on higher concentrations of sugars, you can dilute the sample (just a other way around). The thumb rule of carbohydrate inhibiton test is that you should bring down the titer value to 8 (preferable) because the aim of the assay is to determine the minimum concentration of sugar that is needed to completely inhibit the heamagglutination of just a single, two or three well titer value that is predetermined. The reason to insist sample dilution is that concentration of sample is not a concern in this assay (unlike haemagglution assay) as all it needs to show is mere qualitative agglutination prior to the inhibition assay. To calculate the dilution fold for sample, let's say you have a titer value of 32 (5 wells) for sample, make a reverse titer count from 5th well to 3rd well in order to achieve the 8 titer value i.e 2 for 5th well, 4 for 4th well and 8 for 3rd well. The number 8 which came for 3rd well is your dilution fold (8 fold dilution).
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