How to coat and block the ELISA plate

Today were going to talk about how to prepare the Elisa plate for samples. In order to really understand whats going on here. Heres a little schematic youre going to start with an empty plate which is special because it binds proteins with high affinity and so the first thing youre going to do is adding a solution of capture antibodies to the empty wait wells of a well plate. Over time, those antibodies are going to adhere to the surface of the plate. After coating the Elisa plate with the capture antibodies theyre still going to be regions in the surface of the Elisa plate that can bind protein. Basically we need to empty spaces between the capture antibodies. 

So the next step is going to be blocking the plate which essentially is trying to cover up any remaining available space between the capture antibody thats coated to the plate. So basically blocking reduces the background signal in your Elisa, so lets just jump right into it and discuss how we do this. The first thing you want to do is spraying your surface with ethanol. To wipe away any dust, and just wipe it with a kimwipe or a paper towel to make sure that youre working on a clean surface and then after that youre going to prepare all of your materials and reagents. The materials that youre going to need include your pipette tips, an empty reservoir, the 96-wells Elisa plate, some DP BS or PBS will work and youre going to need a vial containing an aliquot of capture antibody. So what youre going to do first is using your pipettor mix a few times the capture antibody aliquot to make sure that the antibody is distributed and then place the droplet onto the empty reservoir. So for the next step, youre going to add ten milliliters of PBS to the reagent reservoir and its important to note that you dilute capture antibody in PBS because youre trying to maximize binding to the Elisa plate. 

After youve diluted the capture antibody in PBS, what youre going to do is setting your micro pipettor to 100 microliters and pipette back and forth to mix the capture antibody thoroughly and once you feel that its well mixed, carefully take 100 microliters without getting any bubbles and start pipetting it into each well of the 96 wells plate. Now youre just gonna have to keep going back and forth until you finish all the walls of the plate and so jumping right ahead to the last one. This is usually the trickiest one because youre almost out of reagents, so you have to be careful not to get any bubbles but once you feel satisfied that you dont have any bubbles just add it. So after that, just give the plate a gentle tap to remove any air pockets. After that, wrap the plate in some plastic wrap in order to prevent any reagents from evaporating overnight when you leave the plate in the refrigerator and after its been wrapped thoroughly, just grab a post-it note or piece of tape, so that you can label the plate in order to prevent yourself from forgetting what step youre on or to prevent other people from picking up your plate and using it for something else. 

So again, youre going to leave your plate now overnight in the refrigerator at 4 degrees Celsius and what will happen is the capture antibodies will adhere to the plate and some will remain in the solution. The next day were going to wash our plate three times with PBS in order to remove any unbound cap antibody from the wells and the Elisa plate. So in order to do this, youre gonna need some wash buffer which is just PBS with a little bit of detergent and fill up your reservoir and then take your 96 well plate and with a kind of a forceful thrust just empty out the contents into the sink and blot it against a paper towel. I like to wipe away any residual drops with a kimwipe so that I dont get my clubs wet and then take your micro pipe, better your multi-channel micropipette, set it to 200 microliters and just start by filling the wash buffer into each well of the Elisa plate, so 200 microliters should be more than sufficient to fill up the plate. 

Once youre in done pipetting, I like to just give the plate sort of a gentle tap to make sure everythings mixed and then repeat the emptying step and repeat filling the plate and empty, youre gonna do this a total of three times in order to make sure that youve removed any residual capture antibody from the well and this process of washing and emptying is gonna be repeated throughout the process of Elisa. Were just gonna be telling you that you have to wash after this. 

After having washed away all of the residual capture antibody from the Elisa plate, the final step is to add the blocking buffer and so the nice thing about Elisa is all these steps are a little bit repetitive, so just like we put 10 milliliters of PBS into the reservoir, you just kind of take blocking buffer and put it in the reservoir and pipette into each well and then youre going to wrap it in plastic wrap and you can put it in the refrigerator overnight or you have the option of leaving it at room temperature for two hours and this is sufficient to allow the blocking buffer to fill up any remaining space between the capture antibody. After the two hours or overnight incubation, you are going to simply wash out the blocking buffer. Once youve washed away all of the blocking buffer with the washing buffer three times as we showed previously, you are all set and ready to add your samples and proceed with the Elisa.