What Are the Steps of the ELISA Technique?
The concept of ELISA technique is essential in biology and helps explain real-world biological processes and exam-level questions effectively.
Understanding ELISA Technique
ELISA technique, or Enzyme-Linked Immunosorbent Assay, is a widely used laboratory method for detecting and quantifying specific proteins, antibodies, hormones, or antigens in a sample. This immunoassay technique relies on antigen-antibody interaction, an important concept in immunity, antibodies, and antigen research. The ELISA test enables the diagnosis of diseases such as HIV, dengue, and COVID-19 by measuring molecules in blood or other body fluids.
Principle of ELISA Technique
The ELISA technique is based on the specific binding between an antigen and its corresponding antibody. In ELISA, either the antigen or antibody is attached to a solid surface (such as the wells in a microtitre plate). An enzyme-linked antibody or antigen is then added. If the target molecule is present in the sample, the antibody-antigen reaction occurs, forming a complex. After adding a substrate specific for the enzyme, a detectable color change is produced, indicating the presence and often the amount of the target substance.
Steps of ELISA Technique
The ELISA test procedure can be summarized in these main steps:
- Coating: Attach known antigen or antibody to the surface of the wells.
- Blocking: Add a blocking solution to prevent non-specific binding.
- Incubation: Add the test sample containing unknown antibody or antigen.
- Washing: Remove unbound substances by washing the wells.
- Add Enzyme-Linked Antibody: Introduce a secondary antibody or antigen conjugated with an enzyme.
- Substrate Addition: Add a substrate for the enzyme. If the target molecule is present, a color change occurs.
- Result Interpretation: Measure the color intensity using a spectrophotometer or by visual inspection for qualitative results.
Types of ELISA Technique
There are four main types of ELISA technique based on the method of antigen-antibody binding and detection:
| Type | Description | Example Use |
|---|---|---|
| Direct ELISA | Uses one enzyme-linked antibody that binds directly to the target antigen in the sample. | Detecting antigens in food tests |
| Indirect ELISA | Uses a primary antibody and an enzyme-linked secondary antibody for detection. | HIV antibody detection |
| Sandwich ELISA | "Sandwiches" the antigen between two antibodies. Very specific and sensitive. | Pregnancy hormone (hCG) test |
| Competitive ELISA | Sample antigen competes with labeled antigen for antibody binding; signal decreases as sample increases. | Detection of drugs or small molecules |
Applications of the ELISA Technique
The ELISA technique is vital in diagnostics, research, and industry:
- Detection of viral and bacterial infections (e.g., virus infections, HIV, Hepatitis, Covid-19)
- Measurement of hormones and proteins (e.g., human insulin)
- Screening of allergens in food science
- Detection of antibodies after vaccination
- Monitoring immune response in microbiology and medical labs
Advantages and Limitations of ELISA Technique
- Highly specific and sensitive
- Quantitative as well as qualitative results
- Simple and quick process
- No need for radioactive materials
- But requires proper refrigeration and can be costly to prepare enzyme-linked antibodies
Common Mistakes to Avoid
- Confusing ELISA technique with other tests like western blot or PCR
- Not following correct washing steps, leading to false positives or negatives
- Ignoring the difference between qualitative and quantitative result interpretation
Real-World Applications
The concept of ELISA technique is used in medicine (disease detection), agriculture (food safety), biotechnology (protein quantification), and even forensic science. Vedantu helps students relate the ELISA test to real-world health examples and biological projects.
Worked Example – ELISA Test Process
Let’s understand how a pregnancy hormone (hCG) is detected:
1. Antibody specific for hCG coats the test well.
2. Add patient's urine sample; hCG (if present) binds the antibody.
3. Wash away other substances.
4. Add enzyme-linked antibody to bind the hCG complex.
5. Add substrate; color change means hCG is present—confirming pregnancy.
Final Understanding: The ELISA technique enables accurate, quick diagnosis via simple biochemical reactions.
Practice Questions
- What is the principle of the ELISA technique?
- Describe and compare the four main types of the ELISA technique.
- Explain the step-by-step process of performing an ELISA test.
- List real-life uses of the ELISA technique in healthcare and research.
- Draw and label a diagram of the ELISA process.
In this article, we explored ELISA technique, its key processes, real-life significance, and how to solve questions based on it. To learn more and build confidence, keep practicing with Vedantu.
Related Concepts for ELISA Technique
- Enzymes
- Difference Between Antigen and Antibody
- Active and Passive Immunity
- Recombinant DNA Technology
- Immunity
- Antibodies
- Microbiology
- Antigen
- Virus
- Human Insulin
- Vaccination
FAQs on ELISA Technique Explained: Principle, Procedure & Uses
1. What is the ELISA technique?
The ELISA technique, also called Enzyme-Linked Immunosorbent Assay, is a sensitive immunoassay method used to detect and measure specific antigens, antibodies, proteins, or hormones in blood or other samples. It works on the principle of antigen-antibody binding followed by an enzyme-substrate reaction that produces a measurable color change.
2. What are the 4 steps of ELISA?
The four main steps of ELISA are:
1. Coating: The antigen or antibody is coated on the solid surface of microtitre wells.
2. Binding: The sample containing antibody or antigen is added and binds specifically.
3. Washing: Unbound substances are washed away using wash buffers.
4. Detection: Enzyme-linked secondary antibodies bind and react with substrate to produce a color change indicating presence or quantity.
3. What is the principle of ELISA test method?
The principle of ELISA is based on the specific binding of an antigen to its corresponding antibody. The enzyme-linked antibody binds to the antigen or antibody in the sample, and upon addition of a substrate, the enzyme catalyzes a reaction producing a color change. The intensity of this color is proportional to the amount of antigen or antibody present.
4. Which diseases use the ELISA test?
The ELISA test is widely used to diagnose several infectious diseases such as HIV, COVID-19, Rubella, Dengue, Hepatitis B, and TB. It is also employed in pregnancy testing and detecting food allergens due to its specificity and sensitivity.
5. Who discovered the ELISA technique?
The ELISA technique was developed in the early 1970s by Peter Perlmann and Eva Engvall. Their work revolutionized immunoassays by combining enzyme technology with antibody specificity.
6. Are there different types of ELISA?
Yes, there are four main types of ELISA:
1. Direct ELISA – antigen is detected using enzyme-linked antibody directly.
2. Indirect ELISA – uses a secondary enzyme-linked antibody for detection.
3. Sandwich ELISA – antigen is 'sandwiched' between two antibodies.
4. Competitive ELISA – sample antigen competes with labeled antigen for antibody binding.
Each type serves different diagnostic and research purposes.
7. Why is the color change important in ELISA interpretation?
The color change in ELISA is crucial as it indicates the presence and amount of the target antigen or antibody. The enzyme linked to the antibody reacts with the substrate to produce a color; more color intensity means a higher concentration. This visual or spectrophotometric change allows both qualitative and quantitative analysis.
8. How to avoid errors in ELISA during practical exams?
To avoid errors in ELISA, ensure:
- Proper pipetting and timing.
- Thorough washing to remove unbound reagents.
- Use of fresh and correctly stored enzyme-linked antibodies.
- Avoid cross-contamination by using separate tips.
- Follow incubation and reaction times strictly.
These steps improve the accuracy and reliability of results.
9. Can ELISA be used for plant or animal disease diagnosis in biology projects?
Yes, the ELISA technique can be applied in diagnosing diseases in plants and animals by detecting specific antigens or antibodies. It is widely used in veterinary diagnostics and plant pathology to identify pathogens and immune responses, making it suitable for biology project work.
10. Why do students confuse ELISA with Western blot or PCR?
Students often confuse ELISA with Western blot or PCR because all involve molecular detection but differ fundamentally:
- ELISA detects antigens or antibodies using enzyme-linked reactions.
- Western blot identifies proteins by gel electrophoresis and antibody probing.
- PCR amplifies DNA sequences.
Understanding these differences helps avoid confusion in exams.
11. When will direct vs sandwich ELISA be asked in board exams?
Questions on direct vs sandwich ELISA commonly appear in board exams to test understanding of immunoassay variations. Students should focus on:
- Direct ELISA involves a single enzyme-linked antibody detecting antigen.
- Sandwich ELISA uses two antibodies for increased specificity.
Knowing pros, cons, and applications of each type is beneficial for scoring in exams.
