ELISA | Enzyme linked immonosorbent assay | ELISA test | Types of ELISA | Direct and Indirect ELISA

Summary

This video introduces 'Bio Techniques Explained in Less Than Five Minutes,' focusing on the enzyme-linked immunosorbent assay (ELISA). ELISA is a diagnostic tool used to detect antigens or antibodies, vital for identifying viral diseases like HIV/AIDS. The video explains three ELISA variants: indirect, direct, and competitive, each serving different detection purposes. It illustrates how ELISA works, from setting up reactions in a 96-well plate to reading results through a colorimetric detector, providing qualitative and quantitative insights into antigen or antibody presence.

Takeaways

• 🔬 **ELISA Overview**: ELISA (Enzyme-Linked Immunosorbent Assay) is a colorimetric test used to identify substances like antigens or antibodies.
• 🌈 **Colorimetric Test**: ELISA uses color development reactions to provide qualitative and quantitative information about the presence of antigens or antibodies.
• 🧪 **Diagnostic Applications**: Widely used as a diagnostic test for many viral diseases, including HIV and AIDS.
• 🧫 **96-Well Plate Setup**: The reactions are set up in a 96-well plate and read using a colorimetric machine that measures absorbance at specific wavelengths.
• 📊 **Result Interpretation**: Results are displayed as absorbance versus concentration, indicating the quantity of the substance present.
• 🏥 **Indirect ELISA**: Used to detect antibodies against a known antigen, where the microtiter plate wells are coated with the antigen.
• 🩸 **HIV Detection Example**: Indirect ELISA can be used to detect HIV by identifying antibodies in a patient's serum against HIV viral antigens.
• 🔍 **Sensitivity and Specificity**: The presence or absence of color indicates whether the antibodies (or antigens) are present in the sample.
• 🧬 **Sandwich ELISA**: Used to detect the antigen of interest by using monoclonal antibodies to capture and detect the antigen.
• 📉 **Competitive ELISA**: Provides an understanding of how much antigen is present by competing with known antibodies for binding to the antigen.
• 📈 **Quantitative Analysis**: The intensity of color development in Competitive ELISA indicates the concentration of antigens in the sample.

Q & A

What is the primary purpose of the Enzyme-Linked Immunosorbent Assay (ELISA)?

ELISA is used to identify certain substances like antigens or antibodies. It provides both qualitative and quantitative information about the presence of these substances and is widely used as a diagnostic test for many viral diseases, including HIV and AIDS.

How does ELISA give qualitative and quantitative information about the presence of an antigen or antibody?

ELISA uses a colorimetric test that involves antibodies and a color development reaction. The reactions are set up in a 96-well plate and read by a spectrophotometer, which measures the absorbance at a specific wavelength. The result, displayed as absorbance versus concentration, provides insights into the quantity and presence of the substance.

What are the three variants of ELISA discussed in the script?

The three variants of ELISA discussed are Indirect ELISA, which detects antibodies against a known antigen; Sandwich ELISA, used to detect an antigen of interest; and Competitive ELISA, which helps understand the amount of antigen or antibody present in a sample.

How does Indirect ELISA work to detect antibodies?

In Indirect ELISA, wells of a microtiter plate are coated with a known antigen. The antibody to be tested is added, and if specific to the antigen, it binds to it. Then, an enzyme-linked secondary antibody is added, followed by a substrate that causes color development if the primary antibody is present, indicating the presence of the specific antibody in the sample.

What is the significance of using a 96-well plate in ELISA?

A 96-well plate is used in ELISA to facilitate multiple tests simultaneously, allowing for high-throughput screening. Each well can be coated with different antigens or antibodies, enabling the testing of various samples in a single assay.

Can you explain how Sandwich ELISA is used to detect the presence of an antigen?

In Sandwich ELISA, wells are coated with a monoclonal antibody that binds to the antigen of interest. After adding the patient's serum, another monoclonal antibody coupled with an enzyme is added. Upon adding the substrate, if the enzyme-coupled monoclonal antibody binds to the antigen, a color develops, indicating the presence of the antigen in the sample.

How does Competitive ELISA help in determining the quantity of antigen or antibody in a sample?

In Competitive ELISA, known antibodies are incubated with the antigen present in the sample. If the antigen concentration is high, most antibodies will bind to it, leaving few free to bind to the antigen-coated well surface, resulting in a faint color. Conversely, if the antigen concentration is low, more antibodies will be free to bind to the well surface, leading to a darker color, indicating a lower antigen concentration.

What is the role of the enzyme-linked secondary antibody in ELISA?

The enzyme-linked secondary antibody in ELISA binds to the primary antibody that has attached to the antigen. Once bound, it catalyzes a substrate to produce a colored product, which is then measured for absorbance to determine the presence and quantity of the specific antibody or antigen.

Why is color development in ELISA important?

Color development in ELISA is crucial as it provides a visual and measurable indication of the presence and quantity of the target substance. The intensity of the color correlates with the concentration of the antigen or antibody, allowing for both qualitative and quantitative analysis.

How does the absorbance reading in ELISA relate to the concentration of the substance being tested?

The absorbance reading in ELISA is directly proportional to the concentration of the substance being tested. A higher absorbance indicates a higher concentration of the antigen or antibody, while a lower absorbance suggests a lower concentration.

Outlines

00:00

🧪 Introduction to ELISA: A Diagnostic Test for Antigens and Antibodies

This paragraph introduces the video series 'Bio Techniques Explained in Less Than Five Minutes' and focuses on the Enzyme-Linked Immunosorbent Assay (ELISA). ELISA is a colorimetric test that uses antibodies and color development reactions to identify substances like antigens or antibodies. It provides both qualitative and quantitative information and is widely used for diagnosing viral diseases, including HIV/AIDS. The test is performed in a 96-well plate and read using a spectrophotometer to measure absorbance at specific wavelengths. The video explains three variants of ELISA: indirect, direct, and competitive, starting with indirect ELISA, which is used to detect antibodies against a known antigen. The process involves coating microtiter plate wells with the antigen, adding the antibody to be tested, and then using an enzyme-linked secondary antibody with a substrate to develop color. The presence of color indicates the presence of the specific antibody in the sample, such as a patient's blood or serum. This technique is crucial for HIV diagnosis, where the presence of antibodies against HIV antigens indicates infection.

05:01

🔬 ELISA Variants: Direct and Competitive Assays for Detecting Antigens

The second paragraph delves into the direct and competitive variants of ELISA. Direct ELISA is used to detect the presence of an antigen by coating microtiter plate wells with monoclonal antibodies and then adding the patient's serum. If the antigen is present, it binds to the monoclonal antibody, and upon adding an enzyme-coupled secondary antibody and substrate, a color develops, indicating the antigen's presence. Competitive ELISA, on the other hand, is used to determine the quantity of antigen in a sample. It involves pre-incubating antibodies with the antigen and then coating the wells with these antigens. If the antigen concentration is high, most antibodies will be occupied and bind to the antigen, leaving few free to bind to the well surface, resulting in a faint color. Conversely, if antigen concentration is low, more antibodies will be free to bind to the well surface, leading to a darker color. This method helps understand the quantity of antigens present in a sample, which is crucial for diagnostic purposes.

Keywords

💡Enzyme Linked Immunosorbent Assay (ELISA)

ELISA is a biochemical technique used for the detection and quantification of antibodies or antigens. In the video, ELISA is the central focus, with the host explaining how it can provide both qualitative and quantitative information about the presence of substances like antigens or antibodies. It is widely used as a diagnostic test for many viral diseases, including HIV and AIDS, as it can determine whether a person has been infected by identifying specific antibodies in their blood.

💡Antibodies

Antibodies are proteins produced by the immune system to identify and neutralize foreign objects like bacteria and viruses. In the context of the video, ELISA is used to detect the presence of antibodies in a patient's serum, which can indicate an immune response to a specific antigen, such as those from the HIV virus. The script mentions how the presence or absence of these antibodies can be used to diagnose whether a person is HIV positive.

💡Antigens

Antigens are substances that trigger an immune response, allowing the immune system to recognize and attack foreign invaders like bacteria and viruses. The video discusses how ELISA can be used to detect specific antigens, which is crucial for diagnosing diseases. For instance, the presence of HIV viral antigens can be identified using ELISA, helping in the diagnosis of HIV infection.

💡Colorimetric Test

A colorimetric test is a type of analytical procedure that involves the measurement of color intensity to determine the concentration of a substance. In the video, the host describes ELISA as a colorimetric test that uses color development reactions to identify certain substances. The color change is a visual indication of the presence and quantity of antigens or antibodies, which is then measured using a spectrophotometer.

💡96-Well Plate

A 96-well plate is a type of microplate commonly used in molecular biology and immunology, consisting of 96 small wells arranged in an 8x12 grid. The video mentions that ELISA reactions are set up in a 96-well plate, which allows for multiple tests to be conducted simultaneously, increasing the efficiency of the process. Each well can be coated with specific antigens, and the color development in each well is indicative of the presence of corresponding antibodies.

💡Indirect ELISA

Indirect ELISA is a variant of the ELISA technique used to detect antibodies against a known antigen. The video explains that in indirect ELISA, wells are coated with the known antigen, and then the antibody to be tested is added. If the antibody is specific to the antigen, it will bind, and the subsequent addition of an enzyme-linked secondary antibody and substrate will result in color development, indicating the presence of the specific antibody in the sample.

💡Monoclonal Antibody

Monoclonal antibodies are antibodies that are identical because they are produced by one cell and are specific to one epitope on an antigen. In the video, the host discusses how monoclonal antibodies can be used in ELISA to detect specific antigens. They are used to coat the wells of the microtiter plate, allowing for the detection of the antigen of interest in a patient's sample.

💡Color Development

Color development in ELISA is a visual signal that indicates a positive result, meaning that the substance of interest (antigen or antibody) is present in the sample. The video describes how the enzyme-linked secondary antibody, when bound to the primary antibody, reacts with a substrate to produce a color. The intensity of the color is proportional to the amount of the substance present, providing a qualitative measure.

💡Quantitative and Qualitative Information

The video emphasizes that ELISA can provide both quantitative and qualitative information. Quantitative information refers to the amount or concentration of the substance present, while qualitative information indicates the presence or absence of the substance. The absorbance measured in ELISA is used to determine the concentration of the substance, providing a quantitative result. The presence of color change gives qualitative confirmation of the substance.

💡Competitive ELISA

Competitive ELISA is a variation of the ELISA technique used to determine the concentration of an unknown antigen by competing it with a known quantity of the same antigen for binding sites on a specific antibody. The video explains how this method is used to understand how much antigen is present in a patient sample. If more antigen is present, fewer antibodies will bind to the antigen-coated surface, resulting in a faint color, indicating a high concentration of antigen.

Highlights

Introduction to the 'Bio Techniques Explained in Less Than Five Minutes' video series.

Explanation of the Enzyme Linked Immunosorbent Assay (ELISA) as a colorimetric test.

ELISA's use of antibodies and color development reaction to identify antigens or antibodies.

The qualitative and quantitative information provided by ELISA on the presence of antigens or antibodies.

Widespread application of ELISA as a diagnostic test for viral diseases, including HIV and AIDS.

Description of the setup of ELISA reactions in a 96-well plate and reading with a colorimetric machine.

How the absorbance measured by the machine translates to quantitative and qualitative results.

Introduction to the three variants of ELISA: indirect, sandwich, and competitive.

Indirect ELISA's purpose to detect antibodies against a known antigen.

Process of indirect ELISA, including coating the microtiter plate with antigen and adding the antibody to be tested.

Use of enzyme-linked secondary antibody and substrate to develop color as a sign of antibody presence.

Application of indirect ELISA in detecting HIV antibodies in a patient's serum.

Explanation of how the presence or absence of color in the ELISA test indicates HIV status.

Sandwich ELISA's use for detecting the presence of an antigen of interest.

Process of sandwich ELISA, including coating with monoclonal antibody, adding patient's serum, and using enzyme-coupled monoclonal antibody.

Competitive ELISA's role in determining the quantity of antigen or antibody in a sample.

How competitive ELISA differentiates between low and high antigen concentrations through color intensity.

Conclusion and call to action for viewers to like, share, and subscribe to the channel.

Transcripts

welcome to my new video series known as

bio techniques explained in less than

five minutes where I explained

biological concepts in less than five

minutes or so so if you haven't yet

subscribe to my channel please subscribe

and hit that subscribe button so today's

topic is enzyme linked immunosorbent

assay or Eliza so this assay is a

colorimetric

test that uses antibodies and color

development reaction to identify as

certain substances like antigens or it

could be also used for detection of

antibodies now Eliza gives a qualitative

and quantitative information about the

presence of an antigen or an antibody

Eliza is widely used as diagnostic test

for many viral diseases including HIV

AIDS now in Eliza the reactions are set

up in a 96-well plate and that is read

in a common metric machines so where

which is a colorimetric detector

basically which measures the absorbance

of specific wavelength and the result is

displayed in a computer screen and the

result is basically absorbance versus

concentration from that we understand

how much we get the quantitative and

qualitative information about how much

quantity of substance is present now

there are three variants of Eliza we

would discuss one by one by one so the

first variant is indirect Eliza which is

used to detect an antibody against a

known antigen so when we know our

antigen we want to understand whether

there is unknown and what antibody binds

to it we would do our indirect Eliza in

this situation wells of the microtiter

plate are coated with known antigen here

the antigens are marked in yellow then

the antibody to be tested is added into

the wells hopefully that if the antibody

is

specific against that antigen it would

bind to that antigen now our

enzyme-linked secondary antibody is

given and while substrate is provided

the enzyme would give rise to product

and that results in color development so

if the color is developed that is an

indirect proof that the sample that we

provided has that specific antibody

against the antigen which is coating the

surface of these 96 for the plate so

that is how we understand whether our

antibody is present in a particular

biological sample example a patient's

blood or a patient serum etc so this

kind of technique is used to detect HIV

so let's say this human is infected by a

HIV virus and have has AIDS so it's a

hiv-positive human being so as for time

for diagnostic tests his blood is

collected and centrifuge to form the

serum so from the from the serum

fraction we could get the antibodies

generated against the HIV virus proteins

or HIV viral antigens so we know if HIV

virus effect that person and the person

is HIV positive so the antibodies would

be produced in his body if we can detect

those antibodies then we would have a

idea that whether the person is infected

or not so definitely in order to

understand that we would do a indirect

laser so we would see if the antigen is

present that means the person is HIV

positive if the antigen

if the antibody is absent the present is

HIV negative now in a lab what would

happen is they would give the patient

serum serum into the well so which is

previously coated by antigens present on

the viral surface so if the serum

contains the antibody against the viral

antigen then it would definitely buy it

in the antigens coated on the surface of

the web and thereby when we put the

secondary antibody enzyme-linked it

would develop a color yeah

upon giving a substrate but on the other

hand side if the patient serum doesn't

have that antibody produced that doesn't

have the antibody produce should that

should be produced in a viral response

then there would be no color reactions

because the secondary antibodies doesn't

bind to a primary antibody hand unable

to give a color reaction so that would

be HIV negative there are other variants

known as sanducci lyza which is used to

detect antigen of interest for example

we wanted to detect an antigen with our

hand each end is present in a patient's

blood sample or not so what we can do we

know that antigen binds to specific

monoclonal antibody so we can coat the

well of the microtiter plate with

monoclonal antibody after that we can

give the patient's serum or patient

blood or anything like bodily fluid like

that and then we can give another

monoclonal antibody coupled with enzyme

and then we put the substrate now if

their second if the monoclonal antibody

coupled with enzyme is binding to that

particular antigen then in a color would

develop that would tell indeed that

antigen is present in the patient sample

or patients blood so that is how we

understand whether antigen is present in

the sample or not whether indirect

eliezer tells us about whether antibody

is present or not now once we know and

the gene is present another question is

that how to reject how much antigen is

present and competitively lanza give us

an understanding about that so you're

going to be competitively Liza you know

you you are you are asking a question

that how much antibody is produced in a

patient sample or huh so definitely how

much antigen is produced in a patient

sample so what you would do you

previously incubate some antibodies

which you know it is produced against

these yellow antigens and then what you

would do would coat a surface of the

well

DCL Oh antigens now if the antigens are

present in ample then all the antibodies

we provide against it would bind and

there would be very less amount of free

antibodies available to bind to these

antigen coated surface so once we give

it so what would happen is most of the

antibody would be enough floating

condition because it cannot bind to the

antibody Co and the antigen quote of the

quills so ultimately faint color would

be developed so there are two

possibilities one is there is a

dark-colored reaction happening that

means there is very I mean if the dark

color is developing the antigen

concentration is very low because as the

antigen is low when we incubate with

antibody few antibodies bind to the

antigen but most of the antibodies are

now free which can in turn burn bind to

the antigens coated onto the surface of

the well and when we provide secondary

antibody it can give rise to a color

reaction but if there is too much of

antigen present then once we provide our

antibody against that antigen almost all

the antibodies are occupying that

antigen and now when we provide the

whole solution into the Eliza will if

there is no more free antibody which can

bind to the surface coated antigens

that's why once we provide the secondary

antibody after you faint color or no

color is developed so that is how we

understand that how much antigen is

present so if it's more antigen then so

if a lot of antigen is present then a

very faint color would be developed in a

competitive in East Eliza but if there

is very less amount of antigen then the

dark color would be developing in the

competitive Eliza so that is all about

Eliza so I hope you enjoyed this video

if you liked my video give it a big

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