Sandwich ELISA assay

Summary

This video tutorial delves into the specifics of sandwich ELISA, a highly sensitive technique for detecting antigens in biological samples. The process involves using two distinct monoclonal antibodies to 'sandwich' the antigen, resulting in a more precise and sensitive detection method compared to direct or indirect ELISA. The tutorial explains the steps of the sandwich ELISA, from fixing the first antibody to the addition of the sample, washing, and the use of a secondary enzyme-linked antibody to produce a colorimetric reaction indicating the presence of the antigen. Advantages include higher sensitivity and the ability to use crude samples without purification, while the challenge lies in the need for a deep understanding of the antigen and the design of specific antibodies for accurate detection.

Takeaways

• 🧪 Sandwich ELISA is a specific branch of ELISA techniques that is more sensitive, being two to five times more sensitive than direct or indirect ELISA.
• 🔍 The purpose of ELISA is to detect antigens, which are indicative of infections or infective agents in the body.
• 📚 Antigens are fragments of bacterial cells or viral particles, and their detection can help identify specific diseases.
• 🤝 In Sandwich ELISA, the antigen is 'sandwiched' between two different antibodies, hence the name.
• 🧬 Two specific monoclonal antibodies are required for Sandwich ELISA, each binding to different epitopes on the same antigen.
• 🛠️ The ELISA process involves using microplates with wells where one antibody is fixed at the bottom, and the antigen is detected through a series of steps.
• 🚿 Washing is a crucial step in ELISA to remove unbound antigens or antibodies, ensuring accurate results.
• 🔬 The detection process uses a primary antibody to bind the antigen and a secondary antibody linked to an enzyme for signal detection.
• 🎨 The addition of a substrate results in a color change, indicating the presence of the antigen of interest in the sample.
• 🚀 Sandwich ELISA offers advantages like higher sensitivity and does not require sample purification, making it more efficient.
• ⚠️ The main disadvantage of Sandwich ELISA is the complexity in designing the experiment, requiring a good understanding of the antigen and the design of two specific antibodies.

Q & A

What is the main focus of the video tutorial?

The video tutorial focuses on explaining the sandwich ELISA technique, a branch of the ELISA technique, which is more sensitive compared to direct or indirect ELISA.

What is the purpose of performing an ELISA process?

The ELISA process is performed for the detection of antigens present in the body, which can indicate the presence of infections or infective agents, thus helping in diagnosing diseases.

Why is the technique called 'sandwich ELISA'?

The technique is called 'sandwich ELISA' because the antigen is trapped between two antibodies, resembling a sandwich with something in the middle and two things on top.

How many antibodies are required in a sandwich ELISA technique?

Two different specific antibodies are required in a sandwich ELISA technique, each binding to a different epitope of the same antigen.

What is the role of the first antibody in the sandwich ELISA technique?

The first antibody, also known as the fixed antibody, is attached to the bottom of the well in the ELISA plate and is specific to bind with the antigen of interest.

What is the purpose of the wash step in sandwich ELISA?

The wash step is crucial to remove any unbound antigens and antibodies, ensuring that only the bound antigens and antibodies contribute to the final result, thus avoiding false positives.

What is the function of the secondary antibody in sandwich ELISA?

The secondary antibody in sandwich ELISA binds to the primary antibody that is attached to the antigen and is linked to an enzyme, which is necessary for the detection process by breaking down a substrate into a colored product.

Why is sandwich ELISA considered more sensitive than direct or indirect ELISA?

Sandwich ELISA is more sensitive because it uses two antibodies to capture the antigen, which increases the signal and allows for detection of the antigen at lower concentrations.

What are the advantages of sandwich ELISA over other types of ELISA?

Sandwich ELISA offers higher sensitivity, does not require sample purification, and is very specific for the target antigen, making it suitable for complex mixtures.

What is a potential disadvantage of sandwich ELISA compared to other types of ELISA?

A potential disadvantage is the difficulty in designing the experiment, as it requires a good understanding of the antigen and the design of two antibodies specific to different epitopes of the antigen.

Outlines

00:00

🧪 Introduction to Sandwich ELISA Technique

This paragraph introduces the concept of Sandwich ELISA, a specific branch of the enzyme-linked immunosorbent assay (ELISA) technique. It emphasizes the sensitivity of Sandwich ELISA, which is two to five times more sensitive than direct or indirect ELISA methods. The paragraph explains that ELISA is used for detecting antigens, which are indicative of infections or foreign substances in the body. The process of Sandwich ELISA involves trapping an antigen between two antibodies, hence the name 'sandwich.' The explanation includes the preparation of monoclonal antibodies that bind to different epitopes of the antigen of interest, which is crucial for the specificity of the test. The paragraph also describes the initial steps of the ELISA process, including the use of microplates with wells that contain the fixed antibody.

05:03

🔍 Detailed Process of Sandwich ELISA

This paragraph delves into the step-by-step process of conducting a Sandwich ELISA. It begins with the addition of the patient's sample, which may or may not contain the specific antigen. The presence of the antigen leads to its binding with the fixed antibody in the well. Following this, a wash step is performed to remove any unbound antigens, ensuring specificity. The next step involves the addition of a tracking antibody, also known as the primary selective antibody, which binds to the antigen. This is followed by the addition of a secondary antibody linked to an enzyme, which binds to the primary antibody. The final step involves the addition of a substrate that, when broken down by the enzyme, produces a colored product. The development of color in the wells indicates the presence of the antigen. The paragraph highlights the importance of wash steps after each stage to prevent false positives and ensure accurate results.

10:05

📊 Advantages and Disadvantages of Sandwich ELISA

The final paragraph discusses the advantages and potential disadvantages of using the Sandwich ELISA technique. The main advantage is its high sensitivity, which makes it a preferred method for detecting even minute quantities of antigens. Additionally, it does not require sample purification, unlike direct or indirect ELISA, which saves time and cost. However, the technique demands a good understanding of the antigen and the design of a pair of antibodies that bind to different epitopes of the antigen. If the epitopes are too similar, the results may not be accurate. Once calibrated, the test can be reliably used for detecting the specific antigen of interest. The paragraph concludes with an invitation for viewers to like and subscribe to the channel for more educational content.

Keywords

💡Sandwich ELISA

Sandwich ELISA is a technique used to detect antigens with high sensitivity. It involves the antigen being 'sandwiched' between two antibodies, one fixed at the bottom of the well and the other added later in the process. This method is more sensitive compared to direct and indirect ELISA, making it highly effective for detecting specific antigens in complex samples.

💡Antigen

An antigen is a molecule or molecular structure, often a protein, that can be recognized by the immune system and triggers an immune response. In the context of this video, antigens are used as markers for detecting infections or diseases. The Sandwich ELISA technique is employed to identify the presence of a specific antigen in a patient's sample.

💡Antibody

An antibody is a protein produced by the immune system that binds specifically to an antigen. In Sandwich ELISA, two specific antibodies are used to bind to different epitopes on the same antigen, creating a 'sandwich' that allows for the detection of the antigen in a sample. The primary antibody binds directly to the antigen, while the secondary antibody, linked to an enzyme, binds to the primary antibody.

💡Epitopes

Epitopes are specific parts of an antigen molecule to which an antibody binds. In Sandwich ELISA, two different epitopes on the same antigen are targeted by two different antibodies, ensuring specific binding and detection. The use of distinct epitopes is crucial for the sensitivity and specificity of the Sandwich ELISA technique.

💡Enzyme-Linked Antibody

An enzyme-linked antibody is an antibody that is attached to an enzyme, which produces a detectable signal, usually a color change, when it reacts with a substrate. In Sandwich ELISA, the secondary antibody is enzyme-linked, allowing for the visualization of the antigen-antibody reaction, which indicates the presence of the antigen in the sample.

💡Primary Antibody

The primary antibody is the first antibody used in the Sandwich ELISA, designed to bind specifically to the target antigen. It attaches directly to the antigen and is crucial for the initial formation of the antigen-antibody complex. In this video, it is referred to as the 'tracking antibody,' which selects or identifies the antigen.

💡Secondary Antibody

The secondary antibody binds to the primary antibody and is conjugated to an enzyme that facilitates the detection of the antigen-antibody complex. In the Sandwich ELISA, this secondary antibody enables the colorimetric detection of the antigen by interacting with a substrate to produce a measurable color change.

💡Wash Step

The wash step is a critical part of the ELISA process where unbound antigens or antibodies are removed from the wells. This step is essential to ensure that only the bound antigens and antibodies are detected, preventing false positives and ensuring accurate results. The wash step is repeated after each binding stage to maintain the specificity of the assay.

💡Sensitivity

Sensitivity refers to the ability of an assay to detect small amounts of a target substance, such as an antigen. Sandwich ELISA is highlighted in the video as being 2 to 5 times more sensitive than direct or indirect ELISA. This high sensitivity makes it particularly useful for detecting low concentrations of antigens in complex biological samples.

💡Substrate

A substrate in the context of ELISA is a chemical that the enzyme-linked to the secondary antibody acts upon to produce a detectable signal, usually a color change. This color change is what indicates the presence of the antigen in the sample, providing a visual confirmation of the antigen-antibody reaction in the Sandwich ELISA process.

Highlights

Introduction to sandwich ELISA as a branch of ELISA technique with higher sensitivity compared to direct or indirect ELISA.

Explanation of the purpose of ELISA for detecting antigens which indicate infections or foreign substances in the body.

Description of antigens as fragments of bacterial or viral particles that can be tagged to specific diseases.

Overview of sandwich ELISA's process where an antigen is trapped between two antibodies, hence the 'sandwich' analogy.

Requirement for two different monoclonal antibodies specific to two different epitopes of the same antigen in sandwich ELISA.

Use of patient serum in ELISA due to its antigen content and the process of designing specific antibodies for antigen detection.

Explanation of the ELISA kit components, including micro plates with wells for antibody binding and antigen detection.

Process of fixing a specific antibody at the beginning of the ELISA process and its specificity to bind with only the target antigen.

Importance of the washing stage in ELISA to remove unbound antigens and ensure specificity of results.

Introduction of the tracking antibody in sandwich ELISA which identifies the antigen after the initial binding.

Role of the secondary antibody linked with an enzyme in the sandwich ELISA process for signal amplification.

Mechanism of color development in ELISA wells indicating the presence or absence of the target antigen.

Advantages of sandwich ELISA including higher sensitivity and no need for sample purification compared to other ELISA types.

Disadvantage of sandwich ELISA being the difficulty in designing the experiment due to the need for specific antibodies for different epitopes.

Practical application of sandwich ELISA in identifying specific antigens of interest in a complex mixture.

The calibration challenge in sandwich ELISA and the importance of proper antibody pairing for accurate results.

Encouragement for viewers to like and subscribe for more educational content on ELISA and related techniques.

Transcripts

back friends welcome to another video

session from shos biology and in this

video tutorial we'll be talking about

sandwich Eliza we have talked about

Eliza as a whole as a technique we've

already talked about it and it's my idea

to just go back there and understand

that process very well because in this

video I'm not going to talk very tiny

details of Eliza I just talk about a

branch of Eliza that is sandwich Eliza

and uh there are different branches also

called direct Eliza indirect Eliza and

stuff but there's a difference between

sandwich Eliza and direct indirect Eliza

sandwich Eliza is uh in this case much

more sensitive compared with direct or

indirect Eliza it's almost two to five

times more sensitive now first things

why we do Eliza Eliza process is done

due to the detection of antigens that

are present in our body you know

antigens are definite indications of

infection or infective agents in our

body antigen means the fragmentized

portion of bacterial cell or viral

particles in our body so if antigen is

present if we can detect a specific

antigen we can tag it with a specific

disease and we understand about that

disease for that person so we detect

diseases using the detection of antigens

using Eliza techniques or there are

other immunological techniques like Ria

and stuff in Sandwich Eliza the idea is

uh the name Sandwich came because the

process steps as we going to study

you'll see in this case antigen is

trapped between two antibodies so it's

something like

this if I draw it in very very basic

drawing for understanding this is the

antibody and it is actually stuck the

antigen is actually this is the antigen

it actually stuck

between two different antibodies so it

just looks like a sandwich something is

in the middle two things on the top so

that's why it's known as sandwich elizer

technique now any elizer technique the

idea is very simple the idea is this is

the antigen okay so patient patient

sample contains antigen mostly we use

patient serum because it contains

antigen so what we know we know specific

antibody that can bind with that antigen

let's say this is the antigen

Zed antigen Zed that we want to find in

patient's blood so we know about the

antigen Zed so we can design a specific

monoclonal antibody that can bind with

the antiz Z so we prepared that

monoclonal antibody this is the antibody

let's name it as antibody Zed which

binds with antigen Z so we can design it

in the lab and if we know about the

stuff that antigen Zed binds with

antibody Zed we can also design another

antibody that is uh that is that can be

a primary antibody or something that

antibody is also another monoc antibody

say Z2 let's say this is Z1 that will

also bind with antigen Zed so here what

we require we are trying to figure out

the presence of a specific antigen this

is antigen Z so in that case we require

two different specific antibody against

two different epitopes of that same

antigen because these two antibodies are

not binding to the same epitope because

if they bound that create problem we

want them to Bound in the opposite

region of the an so we need two separate

epitopes of the antigen where the

antibodies will bound so we need a

specific antigen which we need to figure

out which we need to detect along with

that we require two

antibody that are very specific towards

two different epitopes of that antigen

so once you know that then we ready to

go for the experiment so this is what we

need to know and this is what we create

in the lab and what we create is small

micro small plates those plates contain

Wells grooves if I draw draw this it

can't be drawn in the two dimensional

space like that but the idea is this is

small groups every group and group looks

something uh

like like this one after another there

will be groups group means antibodies

can be bound at the

bottom okay and you can add

antigen let's say here that the same

reaction if we go on this is the antigen

and again you can put some other

antibody there like that so this is how

the things will go on and we have a

series of that chamber that contains

small tiny groups filled with groups and

now what we do we fix a specific

antibody at the beginning this antibody

Z1 we fix it at the very beginning we

called him the fixed

antibody and we also know what what

antigen to detect so so the grooves only

contain the fixed antibody only that's

that's what it looks like the kit looks

looks like now so so say at the very

beginning it will look something like

this okay this is the beginning don't

forget about this Stu this is the

beginning antibody zed1 and we know this

antibody Z1 is specific to bind with

antigen Zed only it will not bind to any

other antigen so if now what we do we

add the

sample at the sample now if our sample

contains two there are two possibilities

it can contain the an

or may not contain the antigen if our

sample contains the antigen it is going

to attach and bind with antibody Zed one

because this is the antigen

Zed okay and then what we do we do a

wash up okay after applying the sample

we do a wash up why wash up is required

wash up is very very important stage

because this washing stage will help us

to remove any Unbound antigens okay and

because the bound antigens will be fixed

with the antibody because antibod is

anchored properly into the slide well or

the slide

basement so wash we wash them off now

let's say antigen is present so rest

Unbound antigens will be washed away now

the scenario is like this then what we

add we add another antibody remember I

told you we require two antibodies we

attach one antibody at the kit and also

we have another antibody in the solution

this antibody we have in the solution

solution and that solution also come

from the

kit okay this antibody solution and this

antibod is known as the tracking

antibody it's also known as the the uh

primary selective select selectable

antibody which selects or identifies the

antigen okay or tracking antibody

whatever you say this is also primary

antibody because you know primary

antibod is work is just to attach to the

antigen that's it primary anti an body

is not going to give you any expression

okay for the expression we need

secondary antibody so this is the thing

this sandwich is prepared then we have

the second solution that also comes from

the kit and that second solution

contains another antibody that is anti-

primary antibody that means that

antibody can interact with this previous

antibody and that antibody is secondary

because that is attached to enzyme

sometimes we can use a primary antibody

which is directly attached to the enzyme

but that is costlier to produce and the

specificity cannot maintain all the time

for that reason we use two different

antibodies for the detection this is

only Target and then we have the

solution two it contains secondary

antibody it's also enzyme linked

antibody okay so this secondary antibody

will come and join this whole process so

let me draw it here here so here we go

the whole process that we know of

antigen

attached the primary

antibody also attached let draw it small

way and now the secondary

antibody and secondary antibodies linked

with

enzyme the enzyme could be different H

rce peroxides or hrp is used generally

as enzymes here now this is the

secondary antibody this is the primary

antibody now this secondary antibody

once bind to this primary antibody with

the enzyme complex then we add some

substrate and they break this substrate

this enzyme will break this substrate

down into a product okay and that

product is

colorful and we get the color so in the

wells if you if you just just watch this

process

now you see development of colors in the

solution and development of colors will

indicate the samples that you use

whether they contains the antigen of

your interest or not okay so ultimately

but the idea or the mechanism behind

this is this that we talked about every

time we do each of the stages we do a

washer middle there's a wash there a

washing stage every time we doing it

because we don't want to keep any

Unbound antigens or antibodies in the

mixture because if antigen antibody even

it's floating in the mixture still it

going it's going to give us some color

we don't want to do that that will be

false negative we only want the fixed

antigens to give us the result only for

that we need wash up stages conut every

after every this stage okay and it will

produce the colored product we get the

idea that is known as sandwich Eliza

because antigen is getting sandwiched

between two antibodies

for the detection of the antigen now if

we talk about the advantages of Sandwich

Eliza over other types of Eliza sandwich

Eliza is much more sensitive compared

with direct or indirect Eliza two to

five times more

sensitive and another important thing

about the sandwich Eliza is that in this

case of Sandwich

Eliza that does not require this whole

process do not require uh in the

purification of the sample that we take

while in direct or indirect elizer we

need to go for some purification of the

sample or Preparation of the sample

phase that will take time as well as

some expense but in this case we don't

need that we just take the crude sample

extract and we use it why we can use

that why we cannot do that for direct or

indirect Eliza because as we are

separating as we are finding the antigen

based on two different antibody ENT

trapment it is very very specific so

even if you have a a complex mixture

with so many different things so many

antigens different types of antigens

with different epitopes but still this

process can be very handy to find you

the specific Target antigen of your

interest but if you use it for any other

type of Eliza it might not so that's the

very important advantage of Sandwich

Eliza that we know of okay but the

disadvantage is the disadvantage is at

the beginning of Designing this

experiment you need to know

you need to have a very good

understanding of the antigen that we

need to separate and also according to

that antigen you need to design a pair

of antibody that will bind to two

different epitopes of that antigen

because if the epitope ranges are same

or very close then it might not end up

with good results so the calibration of

this whole experiment is difficult

except for that once you calibrate the

stuff for a specific antigen you can run

that antigen as many times or samples as

many times as you want to to identify

your antigen of

Interest so that's all about sandwich

Eliza If you like this video please hit

the like button and definitely subscribe

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videos like that the subscription link

is given here as well as in the bottom

so please do subscribe and keep watching

thank

you