ELISA Tutorial 1: How a Direct, Indirect and Sandwich ELISA Works
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- เผยแพร่เมื่อ 14 เม.ย. 2013
- Here, we discuss the concept behind ELISA (Enzyme-Linked Immunosorbent Assay). Three types of ELISA are discussed: the Direct ELISA, Indirect ELISA, and Sandwich ELISA.
Our ELISA tutorials are kindly sponsored by Calbiotech. Click here to see Calbiotech's ELISA kits: protocol-place.com/Calbiotech
This video and other protocols can be found at our website, the "Protocol Place" - protocol-place.com/
A full ELISA protocol can be found at protocol-place.com/assays/sand...
This video is a part of our ELISA Tutorial Series. Here are all of the videos in this series:
ELISA Tutorial 1: Understand How an ELISA Works - • ELISA Tutorial 1: How ...
ELISA Tutorial 2: Coating and Blocking the ELISA Plate - • ELISA Tutorial 2: Coat...
ELISA Tutorial 3: Preparing and Adding Samples to the ELISA Plate - • ELISA Tutorial 3: Prep...
ELISA Tutorial 4: Finishing the Assay (Sandwich ELISA) - • ELISA Tutorial 4: Fini...
ELISA Tutorial 5: Preparing ELISA Data in Excel for Analysis with GraphPad Prism - • ELISA Tutorial 5: Prep...
ELISA Tutorial 6: How to Analyze ELISA Data with GraphPad Prism - • ELISA Tutorial 6: How ...
Competitive ELISA Tutorial 1: How a Competitive ELISA Works - • Competitive ELISA Tuto...
Competitive ELISA Tutorial 2: How to Use Calbiotech's Competitive ELISA Kits - • Competitive ELISA Tuto...
Competitive ELISA Tutorial 3: Analyzing Typical ELISA Data in Excel - • Competitive ELISA Tuto...
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Thanks for watching!
Youssef Farhat
Protocol Place: protocol-place.com
**Check out some of our other tutorials via the links below**
Competitive ELISA Tutorials - - - • Competitive ELISA Tuto...
ELISA Tutorials - - - • ELISA Tutorial 1: How ...
Gelatin Zymography Tutorials - - - • Gelatin Zymography Tut... - วิทยาศาสตร์และเทคโนโลยี
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My professor talked about all these in the class for about 1 hour, and I didn't really get the point. With this video, in just 10 minutes I understood it by heart. Thank you!
Thankyou for speaking english and explaining in such an understandable way
Thanks so much! I understand this stuff much better now! You're one of the few people on youtube who's very good at explaining things clearly. I'm now trying to figure out how they do this with the magnetic beads through something called immunomagnetic reduction.... lots of study ahead.
This was very helpful! Thank you! Now I'm understanding this more than I was.
7:20 - Sandwich explanation starts
please view this for sand witched explanation th-cam.com/video/BysGjlK_Rq4/w-d-xo.html
had no clue about these methods until you explained it! great video!
Really helpful for beginners, much easier to undestand them most videos i wached. Thanks.
i was having a problem to understand this tecnic, now everything is clean in my mind, thanks for the video, this channel is the most useful channel i ever saw in the youtube ^^
This is so simple and easy to be understood. Thanks so much!
A beginner of immunology.
Thank you very much. You really helped me understand the different types of ELISA.
best video i have seen on elisa
Wow, this was really simple. Thank you!!
This is really amazing, thank you!
This is so awesome; thank you so much for this explanation!!
You're video lecture is great.Thankyou. Helps me alot ✌✌
This video just cleared everything up for me thanks so much
This was phenomenal, thank you very much!
Awesome video! Thank you, really helped me with my final exam prep :)
thanks a lot, i understand so much better for my exam
This is SOOO helpful! Thank you!
many videos available just as long with half the info, so good job!
also, videos that are twice as long with the same amount of info... or half as long with a quarter the info!!! :) :) :)
This is such a good video, thank you so much.
Great video! Your videos are very helpful!
you are amazing at teaching. thank you.
Awesome tutorial. Thanks a lot.
Very accurate, direct, to the point and clear.
Question: yousef, can you please make a video on how to validate these biological methods?
awesome. now it is so much easier for me to understand! thanks alot
Great Job on this! Very useful.
nice video bro.
good vibes .
warm greetings from Ecuador
Thanks for the refresher!
That's so wonderful video! Thank you so much!!!!
THANK YOU SO MUCH! This video just saved me!
Fantastic video, very well explained. Keep up the good work Youssef
Thank you for the encouragement! I'm glad you liked the video.
thank you very much, its nice video. its really soo useful to understand the basic concept of ELISA......
thank you so much for this video :)!!! finally, i understood how it works!
me to
This is so helpful thanks a lot!
Great explanations !
and Yes you did clarify how Elisa works clearly. Thankssss... :)
Great job, so sad that you don't have more tutorial videos, for example: for Flow cytometry
Thanks very much for this Video, really helping me for my Antibody+DNA technology exam haha :)
The vedio really helped me ... thanks 😊
you absolute champion!
thankyou so much.. this video helped me alot
You are very welcome!
Where are you from ?? 🤔
I hope the video helped!
A lot !
thank you very helpful
Huge thanks to Youssef!
Great video!!!!
thank you thank you thank you ,, you are the best
that was helpful , thanks
thanks...really help me...
very usefull! thankyou :)
It is a great video. thanks
Great video! Have you seen our animation on the sample preparation for ELISA?
very useful,,,thanks
Very very good
Very interesting
Thank you!
Question about Sandwich Elisa:
Does it matter how long you wait before adding the stop solution?
Great video btw. It has helped me a lot:))
Thanks
Thank y so much #
thanx قشطة يا دوك !
Great 🤩
Thaaank you!!!
thanks
In Sandwisch ELISA, is the capture Ab the same with primary detection Ab (just like in Indirect ELISA)? I just really want to make sure. Thank you so much for such amazing presentation and explanation! :)
Hii, thanks for video. If you still there, please tell me why people use INDIRECT ELISA? DIRECT ELISA more simple...
Can anyone please explain to me, how can I determine which elisa to use to diagnose different types of diseases?
shout out to Mr. Farhat for this video as well as all the students studying this, I know you are somewhere here, just know
You're a fucking legend. Thanks a lot for this playlist.
Awesome
Try watching with 1.25 speed.
was easyer to understand for me.
Thank you. I learned so much from the Video. Can I use it for my seminar in school project ?
Sure, you can!
شكرا يوسف ^_^
oh my god. you re genius! made a dumb such me understand that alot :D
Would you please give concret examples where the Direct ELISA is used ? Thank you
thank uu
Thanks a lot..
You're welcome!
protocolplace Hi, for sandwich ELISA, why no 'capture antibody' is attached to the sides of the well? Thanks
Hi - the capture antibody can bind to all surfaces of the well that have the appropriate charge, including the sides. I just didn't illustrate it binding on the sides for simplicity.
Well put together, just wondering why competitive ELISA is not included?
Just realized you have a separate video for competitive
great great great video thank you
what are differences between direct and sandwich elisa
What type of Elisa IS THE 'FOURTH GENERATION?'
hi, this is very useful, may i ask if the companies dont havethe Kit of my protein of interest, can i use Antiobodies bymyself and how is that done, thnkx
Sure - check out our website for a step-by-step ELISA protocol that you can try with your own antibodies.
Yes, you can buy your own antibodies (Ab) and do an ELISA on your own. Basically, you need to dilute the Ab that you buy to the appropriate concentration (see our Sandwich ELISA Protocol linked in the video description as a starting point, or you can follow the Ab manufacturer's directions if they have any for ELISAs). You must also purchase a secondary Ab that will bind to the primary Ab that you use (for example, use an anti-goat 2ndary Ab if your primary Ab came from a goat). Good luck!
At 3:54, You don't explain why you need to go from clear to blue to yellow instead of just clear to blue. Why don't you just measure the blue? Why do you have to do an extra step to get yellow?
+Anna Heath I am sure it has to do with wavelength detection and the machine you use to measure the levels
+Anna Heath It turns yellow when you add acid ("Stop solution" as it's often called). This step stops the production of blue color, and the pH change also results in a color change from blue to yellow (but the color change isn't really important, you could read whatever color you wanted with a plate reader but it just so happens to be yellow instead of blue because of this step). If you don't stop the production of blue color prior to reading the plate, then samples will continue to produce blue while the plate is being read. So samples read first will be biased towards having less color since they had less time to change color than samples read last by the plate reader, and this will throw off your measurements.
+protocolplace how come instead of adding a stop solution, we don't just have an incubation time to allow the substrate to be catalysed to completion and measure the spec of that
Is it because the enzyme in the antibody can transform multiple substrates subsequently? And isn't irreversibly modified after the first catalysis of substrate?
when is the indirect method or the direct method more favorable than the other in terms of detecting certain proteins? hopefully you are still active in this account since this was posted like 4 years ago haha
So which Elisa method do they use to test for HIV?
Denny Oak Theoretically, they could use any of the methods to test for HIV. Probably more than one method is used, depending on the company that makes the ELISA kits. You would have to do a web search and read about specific HIV ELISA kits to find out what type they are.
+Delta Ozone +Delta Ozone The evolution of HIV detection by ELISA is currently in it's 'fourth generation'.
I won't bore you with the original 1st and 2nd generation ELISA's, you can find that information easily enough.
The third generation was an indirect ELISA that was to detect antibodies in the sample blood and though it uses the same principle this video describes indirect ELISA as ( |-[ANTIGEN]>-[PRIMARY ANTIBODY]>-[SECONDARY ANTIBODY--CHROMAGEN] ) far as virology goes an 'indirect' ELISA detects antibodies to HIV, as opposed to the virus itself.
The fourth generation which is current was a pretty big breakthrough in HIV detection because it was the first to detect antigen produced by HIV directly (p24 antigen).
It does several things by simultaneously running an indirect configuration in the plate well to detect for human antibody to HIV AND a direct combined with sandwich configuration to detect the actual virus (or rather the antigen p24 it produces).
It's very powerful because you can take direct, indirect and sandwich and link them together, you could have several intermediate antibodies that bind to antigen whilst also serving as part of the human antibody bonding 'configuration'.
This test is most important though for two reasons, the first is sensitivity and the second related to sensitivity is the ability to fairly accurately predict the time of infection (thus giving doctor's a lot more valuable information to work with HIV patient's to maximize they're treatment regiment depending on how long they've been infected with the virus).
It's powerful for sensitivity because both p24 detection AND human antibody detection must show positive. This means that can lead to false positive (even if you have a -ve control it can happen if the problem is systemic ... that is you screwed something up very early on in eg. preparing your solutions).
So if you're using a 3rd generation ELISA you need to rely on a positive or negative result on only one detection criteria. Now with 4th gen if you messed up something for the detection of p24 or the human antibody you'll know because only one will turn colour (you can't only have one, so if the other doesn't turn then your results have to be scrapped). (MOST OF THE TIME)*
The other advantage related to dating of infection is that during initial infection p24 levels are extremely high as the virus is at it's most aggressive stage (all those yummy T-cell's all still virgin's to HIV) and until a certain number of weeks into infection there is no human antibody (or at least not enough to quantify).
*So if they see only p24 and are CERTAIN there can be no false positive's they know the patient's infection is only a few weeks old AND shorten the detection window because people with HIV will test negative to a human antibody ELISA (3rd gen and below) for many weeks until the body has produced enough HIV antibodies for quantification. To detect the HIV p24 antigen directly you can detect HIV theoretically immediately after infection, you just have to find p24 in the blood and there will be tonnes in the first few weeks.
The flexibility of simultaneously detecting the HIV antigen and the human antibody means there are countless configurations of dilution series etc. and can look at the relative proportion of p24 to human antibody to get a ROUGH idea of when infection occurred (ROUGH but MUCH more accurate then ever before).
So basically it's indirect, sandwich and direct (to anchor) but they mix the 3 in crazy novel way's to get as much info as possible about the length of infection and the person's 'susceptibility' to becoming immunologic ally compromised over another person.
Some labs still use 3rd gen but the WHO and a bunch of other medical hard hitter's have made it clear that only 4th generation ELISA should be used for a 'medically accurate' detection and generally speaking the medical community refuses to use 3rd gen. These 3rd gen ELISA's are typically found in developing nations where they just don't have the $$$ for the fancy 4th generation AND 3rd gen ELISA are still GOOD, they just provide a fraction of the wealth of knowledge 4th gen HIV ELISA does.
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Nice video but there is no description of the direct ELISA..? :)
Marie Gøtke Take a look at 2:40 - 4:30 in the video... that's the part about Direct ELISA.
6:30, where the blocking agents start
Elisa (11/20/2021, 9:32:30 AM): Hello! My name is Elisa. How can I help you today?
Know basic concept and principle of ELISA.
This just confused me. Dont you wash the well after every step to get rid of the unwanted bits?
Miss Quirkzy That is pretty much true. But you wouldn't wash after the color-producing step, or else you would wash away all the color and have nothing to measure. What exactly was confusing to you?
Why 96 well? wouldnt be 1 well enough for the scan of one protein of interest?
I would assume to test experimental samples alongside controls, and to perform multiple dilutions for each. A lot of procedures are performed in duplicate or triplicate so the number of wells needed starts to get pretty big pretty quickly.
96 well plates enable you to test many samples at the same time, and to test your controls at the same time, as Matt pointed out.
What is the difference between sandwich and direct Elisa
as far as I understood, during sandwich ELISA only the antibodies that "capture" your desired protein are able to bind to the surface of the plate well. This leads to the other proteins of the sample being washed away, while at the same time only the desired protein binds to the "capturing" antibody and is sandwiched by the antibody that causes the color change.
all this leads to a much higher sensitivity
all this talk of sandwich elisa is making me hungry
svp une video en français !!!
ANHB3316 UWA where u at
Elisa is my real name ;o