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Guest Priya

Queries on Elisa for beginners

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Guest Priya

Hi

I am quite new to ELISA. We have recently purchased few cytokine ELISA kits from mabtech. I have some queries and will appreciate any help on that. 

1. I have high binding plates from costar and also maxisorp strips from nunc. Which is preferred.? Do different plates give different readings. ?

2. Can i store the coated plate. If yes ,how?

3.how long should i give for substrate incubation. When the reaction has to be stopped? Should i do different time points reading and compare or how is it actually done?

4. What is the concentration of stop solution while using tmb substrate?

Kindly clarify. 

Thanks

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Hi Priya,

Welcome to our forum and thanks for your interest in our products. I will try to answer your questions.

1)      The ELISA plates need to be high-protein binding and there are several suppliers available. What plates that are preferred is up to the user. Strip plates are very convenient if you only use a couple of wells for each experiment, but these plates are generally a bit more expensive. Different plates could give different background readings but the difference is generally very minor. If possible (due to the ELISA reader), we recommend to read a reference wavelength (e.g. 650nm), which corresponds to the background from the plastic, subtract the reference OD values from the samples OD reading.

2)      For best results, we recommend to coat the plates overnight at +4 degrees. It is possible to store the plates for some more hours but longer periods are not recommended, since this could affect the antibodies. I cannot give an exact time for how long it is possible to have the plates coated and stored before running the assay, this is dependent also on the specific antibodies. When I do freshly coated plates and do not have the time to do the analysis on them after the overnight incubation, I block the plates and put it back into the fridge. I have had plates in the fridge for about 72 hours (including coating and blocking time), and the assay still works. But please, be aware that this is nothing that we recommend. If you need to store plates you should consider pre-coated plates, they are very stable and can be stored for a very long time.

3)      This is dependent upon what substrate you are using. If you are using SA-ALP you need a substrate that interacts with ALP, e.g. pNPP substrate (available in our webshop 3652-P10), this substrate requires around 45 to 60 min of developing time, it is possible to do as you write to test different time-points, again this is dependent on your study. pNPP substrate does not need to be stopped and should be read at 405 nm. If you instead are using SA-HRP you will need to have a substrate that interacts with HRP, e.g. TMB substrate (available in our webshop 3652-F10). The development time for TMB substrate is between 10-15 min and it should be stopped with e.g. sulfuric acid and directly read at 450 nm.

4)      Concentration of stop solution is dependent upon stop solution used, we usually use sulfuric acid at a concentration of 0.2M.

It seems like you are using our ELISA development kits, this is an adaptable and flexible kit that makes it possible for you to set up the assay in your own way. If you are new to ELISA I think you should consider to start with our ELISAPRO kits, these are complete kits with pre-coated plates and everything included for a straightforward assay. Read more here about our different ELISA formats: https://www.mabtech.com/knowledge-center/product-guide/elisa-kits

This is another link where we describe the ELISA assay: https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-principle on the following pages you can also read about how to determine the sample concentration and other ELISA guidelines.

Please let me know how things go and if you need any more assistance!

Best regards,

Lena Beckman

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Guest Priya

Hi Lena

Thanks for the detailed reply. Ya we have purchased developmental elisa kits. I wanted to get these doubts clarified before i start the work. Will surely get back to you when needed. Thank you.

Priya

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Guest Priya

Hi Lena,

My query is about Reconstitution and dilution of the standards.

1. In the tutorial on the preparation of the standard curve,  the dilutions are prepared from 10000pg/ml and the curve is shown from 2000pg/ml to 3.9pg/ml. As the detection range is 2-200pg/ml, can I use only 250pg/ml -3.9pg/ml for the standard curve preparation or should it be used from 2000 to 3.9?

2. The protocol specifies the standards which are aliquoted should not be refrozen after initial use. As  10ul  is being used to make 10000pg/ml, should I aliquot them with a volume of 10ul in each vial as i cannot refreeze them? And also it is stated that dilutions should be made only 30min before the experiment. If I make a stock of 10000pg/ml, after using 200ul to make further dilutions (as shown in the tutorial), can I freeze (-20degrees) the remaining 800ul and use it later or not? 

3. What about the dilution of other components like monoclonal antibody, biotinylated secondary ab and conjugates. Can we use store at 4degrees the diluted ones and reuse? Is it recommended.

Please clarify.

Priya

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Hi Priya!

You are using the ELISA development kits, they are flexible kits that the researcher can adjust for its own needs. In the protocols for the development kits we leave several decisions to the researcher, this can feel a bit puzzling for beginners, and this is why ELISAPRO kits can be an option. But our development kits are very appreciated by our customers and when you have optimized it and get it to work it is a very price effective assay. Therefore, it is so wise that you ask these questions before starting the assay.

1)      For every analyte ELISA-system we validate the standard and set a standard range. A standard range is the range in which determination of analyte concentration can be done with precision, accuracy and linearity. So different ELISAs can have different standard ranges. The turtorial (on our website) should only be used as an example. You will need to look in the datasheet for the specific ELISA to see the standard range for that specific analyte. If, for example, the standard range is between 1-1000 pg/ml, you need to prepare the standard points/dilutions so that they cover this range. The amount of standard in the standard vial is written in the datasheet. If (again only as example, you need to look in the specific datasheet), the standard vial contains 1ug, reconstitute the standard with for example 1ml of reconstitution buffer (what to reconstitute the standard with is also written in the datasheet/protocol). In this example you will have a standard stock solution of 1ug/ml. Then to a serial dilution of this standard. Exactly the length of the standard curve or the individual steps between the dilutions doesn’t really matter as long as you cover the standard range. It is preferably if the standard range consists of at least 5 points. I include a picture of how to do a serial dilution of a standard that covers the example of a standard range between 1-1000pg/ml. As you can see in the picture, the standard curve covers the standard range and actually go beyond the range. But if you only want to use the points that are within the standard range you can just exclude the 3160pg/ml (used in this example). The different standard point will get different OD values that are plotted in the standard curve. When you look at the OD values of your samples, they should have OD values that are within the OD values of the standard range.

2)      Reconstitute the standard and aliquote the stock solution. If you are using 10ul for each assay I would add more than 10ul per aligoute, at least around 15-25ul (dependent on the tube) since it will be hard to get out every single drop from each tube. These aliqoutes can be stored in the freeze, but when you have thawed one tube it should not be refrozen, and preparations of the standard (further dilutions) should not be frozen and kept for later analysis.

3)      No, it is not recommended to store the diluted antibodies/conjugates. Dilute what is needed in that step of the ELISA. (Example dilute detection antibody in 12ml ELISA diluent (enough for one plate) just before you wash away the samples.)

I really hope this answer your question, and please continue asking!

Best regards,

Lena

hIFNg dilution image standard curve.png

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Guest Priya

Hi Lena,

1.My standard range as per the datasheet is 2-200, 3-300,2.5-250, 5-1000 pg/ml for different cytokines. As you have mentioned that atleast 5 points are recommended,I guess the dilutions which you have provided in the previous reply can be used for all my standard curves as it covers all my standard range. Is that sufficient ?

2. When I was going through the data sheet I found the blocking buffer and incubation buffer are not common to all the cytokines. For eg. Blocking buffer for IL1 has 0.1%BSA whereas for IL4 it is 1% BSA.so I should stick to what is given there right??

3.Not to refreeze doesnt apply to other components i guess as they are just refrigerated. Eg. If I aliquote the antibodies say I have got 80ul of detection antibody which is aliquoted in 4x20ul to avoid repeated thawing I can go back to the same vial right. 

Though they are very simple queries i just wanted to be sure so that I dont end up doing it in a wrong way. 

Awaiting your response.

Priya

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Hi Priya!

1)      Yes, the dilutions provided in the above image can be used for all your standard curves.

2)      For almost all of our ELISA systems a blocking buffer containing 0.1% BSA is sufficient. But for some systems, e.g. Human IL4 ELISA, a higher BSA concentration is needed. Therefore, we recommend a blocking buffer with 1% BSA for human IL4 ELISA. The blocking buffer with 1%BSA can be used for all systems but this high concentration of 1%BSA is not necessary for IL1alpha nor IL1beta.

3)      The standard should be reconstituted, aliquoted and frozen. Do not re-freeze thawed aliquots of the standard. There is no need of aliquoting the other components, they can all be kept in the fridge for 18months (date from delivery). Take out the antibody vial from the fridge, pipette what is needed and put the vial back in the fridge. Always use new and clean tips.

 

Best regards,

Lena

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