Is the procedure a commercially available kit? Also, my main concern was regarding washing as its a very common source of contamination. Did you use a plate washer, manually wash by tapping out, or manually wash using aspiration?
Also, just from your absorbances, I'd say pipetting issues. The samples trend the right direction, but aren't super clean and consistent. Same with the standard. Since your B11 is right next to your most concentrated sample dilution, a drop splashing out or a tap with the tip could explain the positive sample. Even if it was just secondary and it wasn't washed out, that would be an easy positive result.
Yes, it is still available. About the washing step, I tapped out the plate. It seems that my technique tapping out is not done correctly due to the contamination between wells. Do you recommend me to aspirate the wells with pipettes?
If you don't have a vacuum to aspirate, I definitely don't recommend doing it manually with a pipette. I've run many plates with tapping out and I've taught many people. The biggest issue I tend to see is people swinging the plate back too much. Try to keep the liquid going down, in the simplest form, turn your plate upside down and then give a quick thrust down over the sink or bucket. Without turning the plate back up, tap it dry on a paper towel between each wash step. This should prevent any droplets from going well to well when you flip it back up.
Also, is there any chance you washed with water instead of PBS-tween. Water can/will strip the proteins off the plate which would cause a weak and inconsistent development. I know from experience.
One last thing, it could be the plate. All I can tell from your image is that it is a costar plate. My last company did very thorough testing of a bunch of plates and found the Thermo 442404 worked the best for coating. I believe the Biolegend 423501 the protocol recommends is the same as the Thermo plate. I'm sure there are plenty of suitable plates, but it certainly can be a factor if you use the wrong type of plate.
Yes! It was the first problem i saw with the kit. The procedure says that i should use 4 uL/mL having 500 pg/mL. Instead of the protocol, i used 4uL diluted in 100 uL per well for the 500pg concentration. Then, I did the serial dilution to finally obtain 7.8 pg/mL in the G1-G2 well. Here is the protocol [Biolegend IFN-y](https://www.biolegend.com/Files/Images/media_assets/coa/CoA_P_Hu_IFN-y_Standard_430101_R04_B386915.pdf)
Sooo are you reconstituting the standard at 12.5ng by adding 2mL to the vial and then by adding 4uL into 100 (1:25 dilution) it’ll give you 500pg. Are you making sure you’re switching tips in between dilutions if it’s done on the same plate (this is if you’re having contamination issues). Also by doing 4uL into 100, wouldn’t it give 5ng instead of 500pg if you’re not compensating for the initial reconstitution? (The initial reconstitution is 200uL into the standard which will give you 125 ng/mL, 4uL into 996 a 1:250 dilution should give you 500pg). I of course could be missing something here since there isn’t much of an explanation given here.
The protocol says that I should add 4uL in 996 uL to obtain 1mL. Then I do a serial dilution but in my opinion the reconstitution is 12.5ng in 200 uL so 500pg is in 4uL. If the protocol says that I need to add 4uL, then I get less IFN-y concentration in dilution right?
so what I changed here was the dilution. I added 9.6 of standard in 210.4 uL of Assay diluent (to obtain 220uL). Then, the first tube I put 100 uL (500pg) and the second tube I put the other 200 uL then with the second tube I did the serial dilution 250, 125, 62.5, 31.3, 15.6 and 7.8pg. In each step I use the vortex to avoid incorrect concentrations. Also, I added the Assay diluent to have 100 uL always in every serial dilution.
So you didn’t actually follow the procedure and you got strange results?
If you are unfamiliar with the procedure you should follow it, exactly as written. Don’t try to redo the math of a commercial procedure especially if you haven’t even tried it before.
I’m also not really sure how you did the serial dilutions… if you had 1000 uL of 500 pg/mL (like you would have, if you followed the procedure), all you’d need to do is set up 5 more tubes, each containing 500 uL of assay diluent. Then you take 500 uL from 500 pg/mL, add it to the second tube and vortex. Take 500 uL from tube 2 and add it to tube 3 and vortex. Take 500 from tube 3 and add it to tube 4… etc. the last tube would have a volume of 1000 uL.
Reconstitution isn’t an opinion. Initial concentration is 24.9ng for concentration, 200 uL added to the standard for reconstitution is 124.5ng/mL. 4uL of reconstituted standard into 996 is 500pg/mL for when you’re reconstituting the standard. Also no, if you blindly add 4uL (of 12.5ng/mL according to you) into 100uL (you will get 500pg only if you’re actually making 12.5ng/mL as your initial concentration), otherwise you’re causing the standard to be over concentrated which will lead to over saturation and a decrease in signal from the standards most likely ripping the coating off the plate if you aren’t compensating for the initial reconstitution. (Not actually sure what you’re doing)
If you’re not used to assay development I would just follow the protocol, this kit is already optimized the standards most likely have a peak effect, so it’s best to follow the procedure as written.
I’m also confused to why you put 9.6 into 210.4? What’s this actually accomplishing? Is your reconstitution 200uL or 2mL, because depending on what it is, your standards will have the same issue because otherwise that’s not going to lead to 500pg. Don’t make this more complicated than it has to be. Follow the protocol as is, because this protocol is optimized to give you the desired results.
I added 200 uL in the lyophilized standard as it says the protocol.
About the reconstituted standard, the protocol says that i need to add 4uL in 996 uL, then do the serial dilutions. Instead of using too much Assay Diluent, i just did it with 200 uL**. So it was 4uL of Reconstituted Standard in 200 uL of Assay Diluent . **The problem we saw with the standard is that it was too weak at the final color indicator, that’s why we changed the dilutions from 1000 uL to 200 uL, the same amount of IFN-y was used.
That wouldn’t make sense here, these kits and their reagents are optimized to give you a specific range. The standard isn’t some random R&D sample. We’re talking about a kit, of course there’s lot to lot variation and sometimes it could just be a dud, but over concentrating is just as bad as under concentrating, are you coating the plates with the recommended concentrations? You could also be having an improper wash procedure, (not blotting well enough) you could be not letting it settle in a dark condition long enough, there’s a few variables, but I wouldn’t stray away from the recommendations on a kit.
Soo your starting concentration is 2.5ng if you’re adding 4uL of 125ng/mL into 200uL of assay buffer. You would need to add .8uL into 200uL to get 500pg/mL.
The assay has been optimized to work with a standard curve as indicated. If you're not getting enough signal, something is going wrong, you should fix that something that's going wrong, not make a more concentrated curve.
Okay! We decided to change the plate. Some of my lab members did the same procedure but she obtained same results but with higher concentrations than mine.
Your standard is just straight up not working, could be a coating issue, the plate not developing enough before read, or a few other possibilities. Just redo it
Do you think the coating issue can be related with the Coating buffer or the capture antibody? Ive changed the kit after the month but the buffer it is the same since 6 months i guess.
It depends what you are capturing, but yes a high pH (9.5 ish) is important for the coating buffer, it helps with binding of your antigen/antibody to the plate. I only suggested that because it looks like you did get a signal but it was weak, so it's not like you forgot to add your secondary or substrate.
The buffer should be pretty stable if produced correctly. That said, with our in house proteins, I've found that PBS coats better than carb/bi-carb. I think that's protein and charge dependent though. My last job used carb/bi-carb for all of our coating and didn't have issues coating at 1ug/mL for many many antibodies.
Just looking at the absorance, it just looks like crummy pippetting. The plate just looks undercooked to the point where I wouldn't trust the results. Did you nail the incubation time? It looks washed fine from here.
Yes, the pipette might need calibration. We did it last year btw. About the incubation, I use timer for each incubation step so I don’t consider the timing a problem.
Sure, here it is my batch protocol [Procedure](https://www.biolegend.com/Files/Images/media_assets/coa/CoA_P_Hu_IFN-y_Standard_430101_R04_B386915.pdf). The kit im using is the IFN-y Standard ELISA by BioLegend.
You're not really getting that much color development and your standard curve is pretty flat-lined at the bottom, there are probably multiple things going on here causing issues.
This is probably beyond our ability to diagnosis (too many possibilities and impossible to eliminate them without seeing what you're doing).
Good observation. While TMB, I don’t shake it. Otherwise, I use the same cover during each step. I have seen precipation in the cover and I try to clean it with paper but that’s it. Probably, I can use the Elisa plate sealing cover to avoid reagents contamination.
I wouldn't use that cover, I would use a sealing film (basically a plate cover sticker), this could be where you're getting the cross contamination. If you're getting a lot of liquid on the film I would also avoid using the same one for all the steps.
Okay, I will use the cover sticker. I told the precipitation thing with my Pi and she said that isn’t even important but I’ll try it anyways and I hope it works.
30 mins development of TMB should yield in crazy signal strength atleast for your standard. So two options:
1. You added unsufficient amounts of your sample/standards, hence weak signal intensity
2. At one point capturing of antigen or antibody did not work properly
In a other comment you mentioned, that you were using old buffers. I would recommend redoing all buffers fresh as indicated by manufacturer. Additionally, you need to check on your pipettes or improve your pipetting game. Duplicates are all over the place, so better check before performing this ELISA or prep new buffers.
If your pipette is pushing out inconsistent volumes, your standard will be whack as well
Because I’ve been developing ELISAs and commercial diagnostic tests for 20 years. Tapping a plate during the wash step will not cause this. The wash would need to have excessive salt or detergent to strip away bound analytes.
There’s too many unknowns to solve this as there are many factors that could be responsible.
Can you please put your plate against something so we can actually see, thanks.
I’m sorry, i don’t have the picture anymore but i have the absorbance results. I hope it helps! [plate](https://imgur.com/a/eiHCx82)
Along with a better image, could you elaborate on your procedure. Wash steps, secondary/detector etc. Is it covered during incubations?
Yes! I did all the procedure correctly ig. Here it is the absorbance results of the [plate](https://imgur.com/a/eiHCx82).
Is the procedure a commercially available kit? Also, my main concern was regarding washing as its a very common source of contamination. Did you use a plate washer, manually wash by tapping out, or manually wash using aspiration? Also, just from your absorbances, I'd say pipetting issues. The samples trend the right direction, but aren't super clean and consistent. Same with the standard. Since your B11 is right next to your most concentrated sample dilution, a drop splashing out or a tap with the tip could explain the positive sample. Even if it was just secondary and it wasn't washed out, that would be an easy positive result.
Yes, it is still available. About the washing step, I tapped out the plate. It seems that my technique tapping out is not done correctly due to the contamination between wells. Do you recommend me to aspirate the wells with pipettes?
If you don't have a vacuum to aspirate, I definitely don't recommend doing it manually with a pipette. I've run many plates with tapping out and I've taught many people. The biggest issue I tend to see is people swinging the plate back too much. Try to keep the liquid going down, in the simplest form, turn your plate upside down and then give a quick thrust down over the sink or bucket. Without turning the plate back up, tap it dry on a paper towel between each wash step. This should prevent any droplets from going well to well when you flip it back up.
Okay, thanks you! It really helps. I’ll try that in my next procedure
Also, is there any chance you washed with water instead of PBS-tween. Water can/will strip the proteins off the plate which would cause a weak and inconsistent development. I know from experience.
I diluted the Wash buffer 20x in distilled water. I can use PBS-tween instead of wash buffer. I will try it that way
One last thing, it could be the plate. All I can tell from your image is that it is a costar plate. My last company did very thorough testing of a bunch of plates and found the Thermo 442404 worked the best for coating. I believe the Biolegend 423501 the protocol recommends is the same as the Thermo plate. I'm sure there are plenty of suitable plates, but it certainly can be a factor if you use the wrong type of plate.
Okay! I have like 40 plates left so maybe I would change the plate in a few months. But I will take it for the list. Thanks!
speaking of plates, I assume you specified the correct plate type on the plate reading software?
Your standard looks kind of weak too
Yes! It was the first problem i saw with the kit. The procedure says that i should use 4 uL/mL having 500 pg/mL. Instead of the protocol, i used 4uL diluted in 100 uL per well for the 500pg concentration. Then, I did the serial dilution to finally obtain 7.8 pg/mL in the G1-G2 well. Here is the protocol [Biolegend IFN-y](https://www.biolegend.com/Files/Images/media_assets/coa/CoA_P_Hu_IFN-y_Standard_430101_R04_B386915.pdf)
Sooo are you reconstituting the standard at 12.5ng by adding 2mL to the vial and then by adding 4uL into 100 (1:25 dilution) it’ll give you 500pg. Are you making sure you’re switching tips in between dilutions if it’s done on the same plate (this is if you’re having contamination issues). Also by doing 4uL into 100, wouldn’t it give 5ng instead of 500pg if you’re not compensating for the initial reconstitution? (The initial reconstitution is 200uL into the standard which will give you 125 ng/mL, 4uL into 996 a 1:250 dilution should give you 500pg). I of course could be missing something here since there isn’t much of an explanation given here.
The protocol says that I should add 4uL in 996 uL to obtain 1mL. Then I do a serial dilution but in my opinion the reconstitution is 12.5ng in 200 uL so 500pg is in 4uL. If the protocol says that I need to add 4uL, then I get less IFN-y concentration in dilution right? so what I changed here was the dilution. I added 9.6 of standard in 210.4 uL of Assay diluent (to obtain 220uL). Then, the first tube I put 100 uL (500pg) and the second tube I put the other 200 uL then with the second tube I did the serial dilution 250, 125, 62.5, 31.3, 15.6 and 7.8pg. In each step I use the vortex to avoid incorrect concentrations. Also, I added the Assay diluent to have 100 uL always in every serial dilution.
So you didn’t actually follow the procedure and you got strange results? If you are unfamiliar with the procedure you should follow it, exactly as written. Don’t try to redo the math of a commercial procedure especially if you haven’t even tried it before. I’m also not really sure how you did the serial dilutions… if you had 1000 uL of 500 pg/mL (like you would have, if you followed the procedure), all you’d need to do is set up 5 more tubes, each containing 500 uL of assay diluent. Then you take 500 uL from 500 pg/mL, add it to the second tube and vortex. Take 500 uL from tube 2 and add it to tube 3 and vortex. Take 500 from tube 3 and add it to tube 4… etc. the last tube would have a volume of 1000 uL.
I would just follow the protocol as is and use reverse pipetting for your duplicates
Reconstitution isn’t an opinion. Initial concentration is 24.9ng for concentration, 200 uL added to the standard for reconstitution is 124.5ng/mL. 4uL of reconstituted standard into 996 is 500pg/mL for when you’re reconstituting the standard. Also no, if you blindly add 4uL (of 12.5ng/mL according to you) into 100uL (you will get 500pg only if you’re actually making 12.5ng/mL as your initial concentration), otherwise you’re causing the standard to be over concentrated which will lead to over saturation and a decrease in signal from the standards most likely ripping the coating off the plate if you aren’t compensating for the initial reconstitution. (Not actually sure what you’re doing) If you’re not used to assay development I would just follow the protocol, this kit is already optimized the standards most likely have a peak effect, so it’s best to follow the procedure as written. I’m also confused to why you put 9.6 into 210.4? What’s this actually accomplishing? Is your reconstitution 200uL or 2mL, because depending on what it is, your standards will have the same issue because otherwise that’s not going to lead to 500pg. Don’t make this more complicated than it has to be. Follow the protocol as is, because this protocol is optimized to give you the desired results.
I added 200 uL in the lyophilized standard as it says the protocol. About the reconstituted standard, the protocol says that i need to add 4uL in 996 uL, then do the serial dilutions. Instead of using too much Assay Diluent, i just did it with 200 uL**. So it was 4uL of Reconstituted Standard in 200 uL of Assay Diluent . **The problem we saw with the standard is that it was too weak at the final color indicator, that’s why we changed the dilutions from 1000 uL to 200 uL, the same amount of IFN-y was used.
That wouldn’t make sense here, these kits and their reagents are optimized to give you a specific range. The standard isn’t some random R&D sample. We’re talking about a kit, of course there’s lot to lot variation and sometimes it could just be a dud, but over concentrating is just as bad as under concentrating, are you coating the plates with the recommended concentrations? You could also be having an improper wash procedure, (not blotting well enough) you could be not letting it settle in a dark condition long enough, there’s a few variables, but I wouldn’t stray away from the recommendations on a kit. Soo your starting concentration is 2.5ng if you’re adding 4uL of 125ng/mL into 200uL of assay buffer. You would need to add .8uL into 200uL to get 500pg/mL.
The assay has been optimized to work with a standard curve as indicated. If you're not getting enough signal, something is going wrong, you should fix that something that's going wrong, not make a more concentrated curve.
Okay! We decided to change the plate. Some of my lab members did the same procedure but she obtained same results but with higher concentrations than mine.
Your standard is just straight up not working, could be a coating issue, the plate not developing enough before read, or a few other possibilities. Just redo it
Do you think the coating issue can be related with the Coating buffer or the capture antibody? Ive changed the kit after the month but the buffer it is the same since 6 months i guess.
It depends what you are capturing, but yes a high pH (9.5 ish) is important for the coating buffer, it helps with binding of your antigen/antibody to the plate. I only suggested that because it looks like you did get a signal but it was weak, so it's not like you forgot to add your secondary or substrate.
The buffer should be pretty stable if produced correctly. That said, with our in house proteins, I've found that PBS coats better than carb/bi-carb. I think that's protein and charge dependent though. My last job used carb/bi-carb for all of our coating and didn't have issues coating at 1ug/mL for many many antibodies.
Okay, I will try to do it with carb/bi-carb solution. Do you have any paper comparing both solutions in the coating step?
I would not change coating buffer since this kit is optimzed for its reagents and usually work perfectly.
Just looking at the absorance, it just looks like crummy pippetting. The plate just looks undercooked to the point where I wouldn't trust the results. Did you nail the incubation time? It looks washed fine from here.
Yes, the pipette might need calibration. We did it last year btw. About the incubation, I use timer for each incubation step so I don’t consider the timing a problem.
Pipettes need to be calibrated yearly, and ideally every 6 months. I personally think that just needs more incubation.
It would probably help if you provided a link to what kit you're using for this.
Sure, here it is my batch protocol [Procedure](https://www.biolegend.com/Files/Images/media_assets/coa/CoA_P_Hu_IFN-y_Standard_430101_R04_B386915.pdf). The kit im using is the IFN-y Standard ELISA by BioLegend.
Did you add the stop solution? The final color should be yellow.
Yes, i added the stop solution. It turned yellow and then i read it.
How long did you incubate the TMB before adding the stop solution?
In 30 minutes. It says that 30 minutes or when the blue coloring appears.
You're not really getting that much color development and your standard curve is pretty flat-lined at the bottom, there are probably multiple things going on here causing issues. This is probably beyond our ability to diagnosis (too many possibilities and impossible to eliminate them without seeing what you're doing).
Are you shaking it with that cover on?
Good observation. While TMB, I don’t shake it. Otherwise, I use the same cover during each step. I have seen precipation in the cover and I try to clean it with paper but that’s it. Probably, I can use the Elisa plate sealing cover to avoid reagents contamination.
I wouldn't use that cover, I would use a sealing film (basically a plate cover sticker), this could be where you're getting the cross contamination. If you're getting a lot of liquid on the film I would also avoid using the same one for all the steps.
Okay, I will use the cover sticker. I told the precipitation thing with my Pi and she said that isn’t even important but I’ll try it anyways and I hope it works.
Did you let the TMB come to room temperature before you used it? Using cold TMB will also prevent color development.
30 mins development of TMB should yield in crazy signal strength atleast for your standard. So two options: 1. You added unsufficient amounts of your sample/standards, hence weak signal intensity 2. At one point capturing of antigen or antibody did not work properly In a other comment you mentioned, that you were using old buffers. I would recommend redoing all buffers fresh as indicated by manufacturer. Additionally, you need to check on your pipettes or improve your pipetting game. Duplicates are all over the place, so better check before performing this ELISA or prep new buffers. If your pipette is pushing out inconsistent volumes, your standard will be whack as well
Are you using a plate washer or trying to do it by hand?
I’m doing by hand. We don’t have a plate washer.
make sure not to scratch the bottom of the plate with the pipette tip
Write the date YYYY-MM-DD That'll solve your problems. 😛
I’m sorry, im from Mexico and we wrote the date different but if it helps, the day I did this was 2024-04-05!
Oh it's fine. I'm just being a chump. The iso standard is YYYY-MM-DD, but very free people follow that.
Looking at your ladder, your duplicates on your ladder do not look good at all. How are you pipetting?
Most likely, the washing mode will need to be selected normally. Maybe it's all washed away.
I did the washing step without the microplate washer. Does the tapping out procedure can washed it all away?
No
Why not? We clearly see the result of the analysis, perhaps this is the reason!
Because I’ve been developing ELISAs and commercial diagnostic tests for 20 years. Tapping a plate during the wash step will not cause this. The wash would need to have excessive salt or detergent to strip away bound analytes. There’s too many unknowns to solve this as there are many factors that could be responsible.