I`ve read some studies using V-bottom plates for avian rbcs and U-bottom for human. I understand human sink slower than avian. But I see the same results using human rbcs in both well types. Streaming occurs in those clearly positive in both well type and complete agglutination ocurrs in those clearly antibody negative
In the V-bottom plates I do see wells containing bigger less defined pellets than don't stream at all and those that steam slightly but don't tear to the bottom of the well when the plate is tilted
What is the way to report the different outcomes and how are the results supposed to vary between U-bottom and V-bottom shaped wells for human type O rbcs
In summary how do I interpret the different patterns exhibited by the RBCs as far as calling antibody negative vs antibody positive
I ran approximately 40-50 plates this year with the U-bottom and results were reproducible. I had 6 different paramyxovirus isolates with 34 different serum samples, and positive samples tended to tear all of the way. The results were hard to call unless the plate was actually tilted and allowed to settle in between at least three times at room temperature. The negative wells always stayed negative. Is your red blood cell incubation step at 4C or room temperature? Human RBC do tend to sink slower (non-nucleated human vs nucleated avian) and require a longer incubation period to agglutinate. Generally if the pellet does not completely tear and stream then it is negative, because there is still some level of agglutination. If you tilt the plate a couple additional times (remember, let them completely settle back down in the plate before tilting again), usually the questionable sample will reveal themselves. Do you have any positive standards that you can compare you new results to? Or a previous SOP?
Thanks a lot guys, thats helped a lot
James, when you say reproducible, do you mean results are comparable between U-bottom and V-bottom plates or just reproducible when you repeat the same samples on the same plate type (i.e. assay variation is reproducible)
I incubate at room temperature for around 50 minutes but don't tilt until the RBC control has completely settled. My positive standard streams and forms a clear tear in the 20, 40 and 80 dilution wells as it should. This is why I presumed i could call my results positive if they tear, despite most publications stating than they don't tilt the plate with human cells, just wait for some kind of halo to appear. I'm not sure what the U-bottom plates show that the V-bottom wouldn't
Sometimes the RBCs appear to button and I would call it positive but they don't stream at all when I tilt, which is why I ned to know if I have to tilt the plate
So just to clarify James:
1. Both V-bottom and U-bottom are comparable so I can continue with the V-bottom using human RBCs
2. I should tilt and forget about not tilting and looking for halos
3. Grading a positive only if it completely streams to hit the bottom rim of the well (tears) when the plate is tilted, if it streams but doesn't form a tear its negative and not a halo - would you agree?
4. Do you think that it matters that you are looking at the paramyxo viruses (which ones are you looking at? )and I'm looking at influenza regarding all this?
Many thanks - looking forward to hearing from you
1. The samples were reproducible in the U-bottom plates. I did not do a comparison between the U and V. The U looked better for my purposes (and was predominantly used in relevant publications), so I stuck with it. If your results look good with the V's and the human RBC, then definitely stick with them.
2. Tilting (in my opinion) was more definitive, and easier to interpret (just because there is a distinction between full and partial agglutination). Try side-by-side comparisons with a couple positive samples, just for observational purposes.
3. Yes. Only if it reaches the bottom rim of the well. This would be complete hemagglutination inhibition.
4. I am using different paramyxoviruses, but the assay is still based on the hemaglutinin presence along with the virus, and the antibody present in the sera. The influenza should be the same along with choice of human RBC. Make sure human RBC are the best choice.
I attached a 2009 paper validating the method, and one of the few that actually shows a figure of complete HI and partial (negative) HI.
I'm really appreciating your help James so if you don't mind I'll bombard you with a few more questions.
,What RBC species do you use?
Would you mind taking a look at the attached HI plate and tell me if you agree with my interpretation, it's v-bottom with type-0 rbcs and type A/California/07/pdm09 virus after tilting the plate
No problem. I use chicken red blood cells, but it seems horse red blood cells (at 1% instead of 0.5%) work well for human sera. Human Type O is fine, I just mentioned the horse RBC because several publications claim they are superior. How many HA units are you using for your assay? I don't see an attachment.
Mark,
I have attached another protocol from the Pandemic Preparedness Team:
Immunology and Pathogenesis Branch Influenza Division Centers for Disease Control and Prevention for the same assay. They use the V-bottom as well.
Hi James
Ppt file attached, let me know what you think. I use the standard 8Hu per 50ul
Thanks for the link, I'll take a look
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