I did the elisa antibody titre at first, which gave me positive results. After repeating the elisa after 4 weeks, I am not getting any signal at all. I repurified my antigen and recoated the elisa plate, but still nothing. What I dont understand is that how I got positive results in the beginning and now nothing?
More information would be needed. How is your ELISA? Are you evaluating the same samples and using the same reagents?
Hi Kajal, could you please give some more information about your immunisation schedule? How much antigen did you use and how many boosts did you performed? For chicken immunisation, you should use 50- 200ug of immunogen and injections should be performed in 3-4 weeks intervals, collecting the bleeds one week after each boost.
Did you perform a booster injection before repeating the ELISA? If not, that may be the reason you antibody titre decay (normally antibody production falls 20 days after a boost).
Hello, thanks for your help. I did three boosts, and immunised the chickens with 50ug of the immunogen each time. I am coating with my recombinant pure protein for 1hr 37C, then overnight at 4C. The next day, i block with bsa-pbs, wash with pbs,then incubate with my antibody i isolated from the eggs for 2hrs, at 37C. I wash with pbs, coat with secondary antibody which is a rabbit anti chicken hrpo for 1hr, rewash. Then incubate with the substrate (abts in citrate phosphate buffer). And I am getting nothing. Not even with the weeks that I had gotten positive earlier.
Which is your sample? Are you using purified chicken antibodies or sera? Which are the concentratiosn you use (that were positive in teh first assay)? How did you keep your antibodies in the interval between ELISAs? And the antigen?
Do you only wash with PBS? No detergent at all? Im thinking in high non-specific background in some of the assays, although this does not explain why the signal dissapeared.
Did you use the same reagents and plates all the time without any change?
Did you run your purified IgY in a SDS-PAGE gel to check that the IgY was correctly isolated from the egg yolks? Maybe you are not getting results in your latest ELISA because there was a problem during the IgY isolation process. You could do an antibody titre using serum dilutions to know if the immunisation protocol is working. That should give you an idea of where the issue is arising from – from the eggs IgY or from the reagents you are using in your ELISA.
Also, if you have any other IgY in your lab, check that your HRP-anti-chicken IgY antibody is working before doing anything else. You can also try using higher antigen concentrations to coat your plates (not sure how much are you using right now).
Thanks so much for your comments. i have not run my IgY samples on a page yet, but I thought they would work because they worked earlier on an elisa. I will try use another igy to see if the hrp-anti chicken if fine. Thank you so much for your comments.
One of my colelagues, Esteban Justo Gutierrez Calzado, has a lot of experience in chicken antibodies. You can contact him vai researchgate.
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